4:5 "See, I have taught you decrees and laws as the Lord my God commanded me, so that you may follow them in the land you are entering to take possession of it..."

Attempt: Doctrine is actually acquired by study through the Holy spirit, meaning that in both cases the Spirit imparts wisdom.

Attempt: Doctrine does actually prove the principles of lower sciences because firstly, when applicable Sacred Doctrine takes authority over any lower science due to its certainty. Secondly, it defends the principles of lower sciences by establishing & studying the one who gives them operation, God- that effects may inform other effects.

Attempt: Sacred doctrine gets truth directly from God, & so any defect, such as borrowing principles, will come only from human defect as means to understand it, rather than from doctrine itself.

"That which is more certain in itself isn't per se so to us. The authority propagating sacred scripture as true far surpasses that of the sciences." * To give an example, our science of sociology for instance may be completely false & based on a fundamental misunderstanding, even if highly unlikely- this is because our human intellects are so very dark, & though authoritative, can't be called a maximum. Now sacred doctrine is literally revealed by the infallible God, which is to say that it's very certain, the authority behind its certainty is much more great. * Certainty doesn't here refer to how confidently one proclaims a truth is, or how demonstrable it seems.

is AI playing a role in this?

I don't get this part what are they trying to say?

why with open AI

do you think this could help us?

I fell like there needs to be more care about what they are doing to the planet

what does this mean?

and this would be perfect for a 'trained with' or 'upskilled with' trust bar.

This would be perfect for a 'trust bar' where you showcase the logos of each society/association/chapter.

This says missing H5P identifier.

The H5P M/C Qs should be as follows: A study published in the Journal of Nutrition analyzed data collected from more than 50,000 adults aged 30 years and older. Researchers found that individuals who snacked less frequently, avoided eating late at night, and consumed their largest meal at breakfast had, on average, a lower body mass index than those who snacked more, ate later in the day and large dinners. What type of study is this? Invitro::Incorrect. *Observational study::Correct! This is an observational study because the researchers observed participants' eating habits and compared them with body mass index (BMI) without assigning people to different eating patterns or controlling the variables. Intervention::Incorrect. Systematic review::Incorrect. Animal study::Incorrect.

Since data is being collected on dietary habits and weight on humans for this study, and there is no treatment, it is an observational study. In the Journal of Nutrition study, what is the relationship between less snacking and lower BMI? *Correlation::Correct! The study found an association between less snacking and a lower BMI. Because it was an observational study, it cannot determine whether snacking less caused the lower BMI. Other factors, such as physical activity, sleep, or overall diet quality, may also have contributed to the relationship. Causation::Incorrect.

In the Journal of Nutrition study, other factors such as physical activity, sleep, socioeconomic position, or stress might also explain the observed results. What are these factors called? Cooperative factors::Incorrect. Confidence factors::Incorrect. *Confounding factors::Correct! Confounding variables are factors that may influence both the exposure (e.g., snacking habits) and the outcome (BMI). Because observational studies cannot control all confounders, they can identify associations but cannot prove that one factor caused the other. Conflicting factors::Incorrect.

In the study described above, researchers found that individuals who snacked less frequently tended to have a lower BMI. What does this relationship represent? A causal relationship demonstrating that reducing snacking directly lowers BMI.::Incorrect. *A correlation indicating that less frequent snacking is associated with lower BMI.::Correct! The findings demonstrate an association between snacking frequency and BMI, but they do not establish that reducing snacking causes weight loss.

A study randomly assigned 2,072 women in early pregnancy to receive either an iron and folic acid supplement or a more comprehensive supplement containing essential fatty acids and 22 vitamins and minerals. Researchers found no differences in pregnancy or childbirth outcomes between the groups. What type of study is this? Cohort study::Incorrect. Case-control study::Incorrect. *Randomized controlled trial::Correct! Participants were randomly assigned to different supplementation interventions, allowing researchers to compare outcomes while reducing the influence of confounding factors. Randomization strengthens the ability to evaluate cause-and-effect relationships. Cross-sectional study::Incorrect.

A single research study is usually sufficient to establish a clear cause-and-effect relationship between diet and health. True::Incorrect. False::Correct! Scientific conclusions are strengthened by consistent findings from multiple well-designed studies. Even randomized controlled trials have limitations, and evidence should be evaluated collectively before establishing dietary recommendations.

In a recent peer-reviewed study, researchers searched the literature for studies of dietary patterns and incidence of diabetes. The authors found 48 relevant studies. Combining the results, they found that following the Mediterranean diet and DASH diet were associated with a lower incidence of diabetes. Eating more vegetables, legumes, fruits, poultry, and fish was associated with lower risk of diabetes. What type of study is this? In vitro::Incorrect. Observational::Incorrect. Intervention::Incorrect. *Systematic review with meta-analysis::Correct! A systematic review uses a structured process to identify and evaluate relevant research. A meta-analysis statistically combines results from multiple studies to estimate an overall effect or association. The strength of the conclusions depends on the quality and design of the included studies. Animal study::Incorrect.

Two studies examine whether a dietary supplement reduces blood pressure. A small observational study finds that people taking the supplement tend to have lower blood pressure. However, a large randomized controlled trial finds no significant difference in blood pressure between participants receiving the supplement and those receiving a placebo. Which conclusion is most scientifically appropriate? The observational study provides stronger evidence because it reflects participants' real-world dietary behaviors.::Incorrect. The supplement has been proven ineffective for everyone because the randomized trial found no significant difference.::Incorrect. The randomized controlled trial generally provides stronger evidence for evaluating the supplement's causal effect, but the findings should be considered alongside the broader body of research.::Correct! Random assignment helps reduce confounding and provides stronger evidence for causal relationships. However, no single study is definitive, and researchers must consider study quality, statistical precision, and consistency with other findings. Both studies provide equally strong evidence because they investigated the same supplement and health outcome.::Incorrect.

Can we change this to 491 kcal and the correct answer from 28 to 27 grams?

Can we move this to the end of this section and combine with the calculation questions there?

This paper is now published https://doi.org/10.1093/nargab/lqag111

We need testimonials before this section :)

When I click a menu, whole menu changes. It's not a major issue but somewhat feels strange as usually a header with menu stays the same across the website.

This reminds that we need to create a linkedIn account.

How about using NIOSH headforms to show "a few cases" for the image below if this section emphasizes limitations of typical design approach?

Another test annotation!

Just a test annotation.

This is just a first test. @Jonathan_Wexler

I thought it was funny that this quote is usually credited to Mark Twain. But the source says neither of them actually said it. The idea goes back to Jonathan Swift, and the wording changed over time as people repeated it. So a quote about misinformation spreading fast is itself a piece of misinformation that spread and mutated.

This is the trailer for one of the sponge bob movies created in 2019. Movies based on popular tv shows tend to go viral. These movies also usually have a plethora of memes created out of the movie.

This article talks about a people becoming a community from a TikTok showing the positive reasons for social media. Just one video become such an impact on the creativity of people and honestly the joy.

This one really resonated with me, because a lot of what I share isn't because I care about it, but it's for how it makes me look to other people. It makes me think a post can spread far without people actually agreeing with it. They just like what sharing it says.

This makes sense, most people when scrolling on any social media platform are less likely to read bulk text, or anything from links. Having visuals that can be interpreted without much brain power does a better job at engaging the viewer.

This is becoming even more of an issue today with 57.4% of the internet being bots.

how about we link to them? and then remember for the pst?

beginning of november

Japanese were able to annex Korea by appealing to collaborationists

Korean nationalism shifted from being uplifted by the civilizing empire to being in competition with Japanese control

Civilizing mission was used to legitimize imperial control and authoritarianism

Collaboration thus became vilified as the Ilchinhoe collaborationist legitimacy failed and mutual cooperation collapsed

Ilchinhoe legitimacy had been shattered due to their support for the Japanese who now went directly against their own interests

As the Ilchinhoe was suppresed, its claims of popular legitimacy through Japanese support were dramatically weakened by Japanese intervention

Ilchinhoe had rather than increase popular control, had simply allowed the Japanese to involve itself in "reforming" Korean governance

Japan would now take the central role in Korean rule

Empire rejected the intervention of local actors in improving Korean government, and the populist Ilchinhoe was pushed out in favor of authoritarian Japanese goivernance

Preservation of collaboration in the face of increased Japanese authoritarianism

Japan's civilizing mission sought full Japanese control of the nation, civilizing mission sought to reform Korea to benefit Japan

Japanese desire for control was thus being presented as greater centralized control

Ilchinhoe thus represented a threat to Japanese control

Japanese protectorate was predicated on Japanese interests and popular supervision of tax administration would jeopardize, Japanese financial controls

Ilchinhoe's advocacy for the people was hurt as the Japanese protectorate refused to acknowledge its positions

Civilizing empire if it protected Korean rights would be an equal sovereign to a Korean sovereign. Sovereignty merely rested on the protection of freedom and welfare

In order to rescue Korea, Japan would take a dual role in helping restore Korean civilization and participating in the Korean polity itself

Sovereignty as based on the sovereigns will not on the manner of the sovereign itself (a sovereign such as Japan could maintain Korean sovereignty if it merely had the will to do so)

Dependent country would remain independent in substance while the civilizing country provided guidance toward eventual total independence

Japanese would help uplift the Korean people and bring them closer toward independence (civilizing mission)

People as participating in government through popular will but with the maintenance of the monarchy

State as existing through the will of the people

Ilchinhoe moral legitimacy collapsed, as Japan rejected its role as a voice of the people, and collaboration collapsed morally due to Japanese colonial status

Ilchinhoe and Japan mutual cooperation only went as far as both their interests overlapped

Populism premised on popular will and its implementation

Popular choice should be followed. Freedom only comes when popular choice is followed

Ilchinhoe as a populist movement which sought popular advancement as its main goal

Japan as a civilizing empire which could bring reform and progress to a backward Korean monarchy

Tonghak religion as deeply humanist and egalitarian

Korea as a hotbed for Chinese-Japanese imperial rivalry

Korean reformist elites saw opportunity in Japan in their reforming mission and therefore led them to engage/collaborate with Japan in search of reform of Korea

Civilizing mission became a way to justify imperial expansion through moralistic and ideological ideals

Shift to formal colony represented a crisis of Empire, as Empire increased their control, as mutual cooperation was tested by local actors

Collaboration in European empires as part of mutual cooperation and bargaining between local actors and European states

Empire provided a framework in societies where identity and occupation was blurred and occupation was normalized

Collaboration as more normalized in societies where foreign domination had been forged historically and identity was blurred and therefore empire could provide the framework for identity

Collaboration became a moral argument when its assistance led to profound moral conflicts and ultimately failed to legitimize itself

Deliberate accommodation is the deliberate and opportunistic assistance to the occupier

Structural accommodation involved the normal function of society even under accommodation (e.g. unconcious collaboration)

Collaborationist as defined by their ability to justify their collaboration to the conquered subjects

Collaboration as legitimization of colonial/imperial rule

Tension between opportunity and moral judgement

Empire provided opportunity to many local actors and thus national self determination was not the only method of freedom and opportunity

Empire as a deeply diverse polity which saw a multitude of interactions for and against the Imperial Order

Immoral Rights signifies a split from the morality of civilizing empires and a move toward popular rule and collective sanctions (self determination)

Protectorate as a method of modernization

Ilchinhoe as collaborationists who supported the Japanese colonization of Korea

I see the year 2015 has been added next to Gmail and yahoo but not on Microsoft why not just put like SPFDD mark and the dates they started existing or you know started being used so that way the 2015 is associated with yahoo they're not the ones with teamwork

Well you don't get the record twice you get the record on a subdomain of a submarine that doesn't exist

Well only if they have the RUF if they don't they don't even get anything they're not even told

Shouldn't we give them a way to check it or take you or else here so they can open them in a new tub

Is it still Denmark one should we be talking about the new Denmark

This sentence sounds weird is there a easier way to explain it just because the words are like repetitive this reason from an address and so on

Para domain or percept domain but not to for each one how do we explain this clearly I assume if we have the animation on screen will be easier to understand based on what we say

Is there a tool or something they can do to figure out where their website is hosted or their DNS is hosted actually

I feel like I usually tongue the second and slur the next two. I think that sounds hip

I like the dn syllable. I think all these rules are too much and too specific to remember and stick to, but I think over time students will learn what is appropriate. I will have to inquire to see what they think to ensure they are thinking and engaged when playing the music

all of these rules can be learned by listening. Would I expect students to remember these rules?

hmm

I like to do this personally, and I think it would help young groups a lot. I wonder how hard this is to implement and learn

I dont think its this simple

I struggle with tongue stops. I dont think theyre necessary, but what are alternatives

I did this in middle and high school with eraybar. Should I set syllables in stone or should I let them express themselves more?

so then how do you get kids to listen

this is very important and one of the roots of the issue

I see what he is saying- but I feel jazz composition is very valuable too

if they are following general guidlines, is it truly authentic?

I was hoping for a definition I can explain to students, even if it takes a long time for it to unfold

¿Cómo los cambios sociales han influenciado la educación?

https://i.imx.to/i/2026/11/09/78gx9v.jpg

Are these our graphs? Must be our graphs not anthropic

Paragraph break.

remove this word

Italic, with a paragraph break afterwards.

Fable 5.2 did not claim

We've trained agents to predict the director's notes, and judged questions of taste on how well they matched the director's pick.

remove

sub-bullet points

We did 67 bounded tests, comparing tasks given to Fable 5.1 in the past week. We controlled for inputs, ensuring that each had access to the same xyz (say anything about the process here)

Which one did?

we are finishing, Reality Games.

, and right away we had Opus 5.5 test

vs

This absolutely is a problem. The change of intention, while sometimes can be funny, usually results in online discourse. Also the change of intention on actual important issues takes away an important message from real world problems. The internet has become a place of endless arguing and a place to go viral. In reality it should be a place that can spread awareness too.

https://i.imx.to/i/2026/11/09/78gx9k.jpg

• Multitasking with terminal AI tools (like Claude Code or Codex) causes "AI brain fry" when branching tasks and parallel threads overwhelm working memory.
• The author tackles this issue by structuring development as a git-backed priority queue:
  • Every ticket is stored as a markdown file committed directly to the repository alongside the code.
  • AI agents autonomously work through tickets in cycles (researching, building, and running tests) while sensitive code changes remain on dedicated branches.
  • Agents never deploy to production; deployment authority remains strictly with the human owner.
• Human review is funneled into a centralized "owner inbox" powered by a single-page Claude artifact regenerated from git each cycle:
  • Decisions and trade-off rulings are submitted via the interface and recorded back into the markdown cards in git.
  • Closing each ticket flushes its context from working memory, enabling single-task focus.

Hacker News Discussion

• Several commenters recommend embracing idle time and hard-limiting active concurrent AI sessions to 1–3 to prevent cognitive overload.
• Participants debate the primary bottleneck in agent workflows, arguing that external processes (compilation, running CI, extensive test suites) slow down progress more than raw model latency.
• Others advocate heavy upfront architectural planning and Jira ticket breakdown, enabling massive parallel agent execution followed by dedicated review phases.
• Commenters raise concerns over long-term knowledge retention and codebase ownership, noting that reviewing AI-generated code leaves developers without an intuitive mental model of the system.
• A strong critique emerged around the writing style of the post itself, with readers flagging telltale AI idioms, sentence structures, and punctuation as off-putting.

Liberation Psychology and empowerment theory work in tandem to create a means and method of communication between populations. From minority to majority populations to identify the ways that the majority is harming said minority. Through this dialogue the class with power can then make an informed means of change or surrendering of some of their power to better accommodate the minority population.

This theory is valuable to social psychologists as a means to identify discrimination by a majority pop to a minority population. So this would be a valuable theory for my capstone as a means to analyze the relationship between Hispanic immigrants working in the agricultural industry.

Kelly's ecological theory makes for a strong foundation for community activism. He identifies the 4 ways to have an effective means to engage your community to resist and change the institutions that may be enaged in state sanctioned violence. I appreciate Kelly identifying that the simple means of gathering and providing space for affected individuals is an effective means of resistance.

• Fragmentacja rynku vs. spójny ekosystem:
  • Porównywanie bezpieczeństwa iOS i Androida wymaga uwzględnienia różnic sprzętowych – Apple kontroluje cały stos technologiczny, podczas gdy ekosystem Androida obejmuje zarówno flagowce z 7-letnim okresem wsparcia (Google Pixel, Samsung Galaxy), jak i tanie urządzenia bez regularnych aktualizacji.
  • Udział rynkowy na świecie wynosi około 69% dla Androida i 31% dla iOS (w Polsce odpowiednio 64% do 36%), co czyni Androida znacznie częstszym celem masowych kampanii cyberprzestępczych.
• Statystyki CVE i błędy zarządzania pamięcią:
  • W 2024 roku Android odnotował 501 podatności CVE, natomiast iOS 317, przy czym wskaźnik ten zależy od natury otwartego kodu Androida oraz wliczania komponentów dostawców trzecich (np. Qualcomm, MediaTek).
  • Obaj producenci kładą nacisk na eliminację błędów pamięci: Google zredukował ich udział w podatnościach Androida z 76% (2019) do poniżej 20% (2025) dzięki implementacji Rusta, podczas gdy Apple wdrożyło domyślne, sprzętowe tagowanie pamięci Memory Integrity Enforcement (MIE) od iPhone'a 17.
• Zero-daye i ataki celowane:
  • W 2025 roku odnotowano 15 zero-dayów wykorzystanych w atakach na urządzenia mobilne, a około 90% łańcuchów wieloetapowych ataków celowało w platformy smartfonowe.
  • Większość zaawansowanych podatności zero-day przypisuje się obecnie komercyjnym twórcom oprogramowania szpiegowskiego (spyware), co sprawia, że włączenie trybów podwyższonej ochrony (Lockdown Mode w iOS, Advanced Protection w Androidzie) staje się standardem dla osób wysokiego ryzyka.
• Czas dostarczania poprawek i adopcja systemów:
  • Czas reakcji na zgłoszenia Project Zero u źródła jest zbliżony (średnio ok. 70–72 dni), jednak kluczową różnicę stanowi proces dystrybucji aktualizacji do użytkowników końcowych.
  • Wskaźnik wdrożenia najnowszego systemu wypada znacznie korzystniej dla Apple (iOS 26 osiągnął 79% udziału w 9 miesięcy po premierze), podczas gdy Android boryka się z opóźnieniami wynikającymi z łańcucha dostawców, nakładek producentów i operatorów.
• Wycena exploitów na czarnym rynku i konkursy hakerskie:
  • Ceny pełnych exploitów typu zero-click sięgają 5–7 mln USD za iOS oraz do 5 mln USD za Androida u czołowych brokerów (np. Crowdfense), co odzwierciedla ogromny poziom trudności przełamania obu systemów i konieczność tworzenia wieloelementowych łańcuchów przez całe zespoły hakerów.
  • Zmagania podczas Pwn2Own potwierdzają wysoki stopień utwardzenia czołowych urządzeń (Google Pixel oraz iPhone), przy czym ataki udają się głównie na modelach ze złożonymi, dodatkowymi modyfikacjami systemowymi.

• Goldman Sachs updated internal guidance in October 2026 to restrict third-party AI coding agents, specifically Anthropic's Claude Code and Microsoft's GitHub Copilot, strictly to software engineers.
• Most bankers and traders have been stripped of access, though quantitative "Strats" who bridge trading, research, and engineering retain permissions.
• The sudden restriction follows high-profile internal praise in July 2026, when CIO Marco Argenti lauded non-technical employees using "vibe coding" to prototype trading tools without waiting for engineering backlogs.
• The shift addresses critical operational, compliance, and security risks:
  • Running autonomous agents directly on finance workstations that store sensitive client records, trading flows, and internal data exposes the firm to data leaks and unvetted code execution.
  • Code generated by non-engineers bypassed standard peer review, rigorous testing, deployment pipelines, and regulatory information barriers (Chinese walls).
  • Unaudited logic errors in models could trigger catastrophic financial losses, compounded by soaring per-token API costs.
• The policy comes at an awkward juncture: Goldman Sachs and Morgan Stanley are lead underwriters on Anthropic's upcoming IPO, marketing shares for a company whose coding agent Goldman largely barred from its own core business users.

Hacker News Discussion

• Commenters attribute Goldman's reversal to Simon Willison's "Lethal Trifecta" security model: granting semi-autonomous agents co-located access to private data, internet connectivity, and execution permissions creates severe risk of prompt-injection exfiltration.
• Several users suspect an unpublicized blunder or near-miss occurred internally, arguing that tier-one financial institutions typically act this aggressively only after an incident threatens regulatory scrutiny or substantial financial loss.
• The community debated architectural isolation and sandboxing:
  • Many argued agents should never run directly on host machines and must instead be segregated inside ephemeral, memory-less virtual machines or isolated cloud sandboxes.
  • Others countered that excessive isolation degrades the seamless user experience and productivity gains that make agentic tools attractive in the first place.
• Multiple participants highlighted the inherent unreliability of LLM instruction-following, noting that models still fail to reliably separate instructions from untrusted data, making "auto-mode" security safeguards insufficient for high-stakes trading floors.

I wonder if there is a specific ratio of p53 to Y220C that can be established for returning functionality?

Heparin is an anticoagulant molecule,

Tm = Temp @ which ~1/2 protein population is unfolded under reversible equilibrium conditions

Hydrogen or Carbon NMR spectroscopy?

A surface crevice on which part of the protein? P53 is a tetramer so it has 4 components when biologically active, so which chain specifically does the mutation occur on?

The paper doesn't really establish the structure of p53 but some quick searching says that it's a transcription factor with a tetrameric conformation that acts as a molecular sensor and activates in response to DNA damage.

This suggests an increase in stability as the mp is higher.

Lower mp is usually correlated to loss of structural stability, typically changes in the inter / intramolecular forces.

What is the n for mutations? This data point is less useful without a reference to the actual values.

5,000 Restful Ways To Survive A Day Without Napping

saca esto

Revisa el formato de esta tabla para que las lineas queden alineadas como estaban antes que estaban todas prolijas

Revisa el formato de esta tabla para que las lineas queden alineadas como estaban antes que estaban todas prolijas

Pon en el apendice el detalle de estas vacantes

Alinea las tablas de esta sección

estas tablas deberian estar en el formato por pais y en el apendice el detalle por persona

Ojo aca solo se tienen que considerar las salidas que sean de esta semana y no se debe poner eso del riesgo

estas tablas deberian estar en el formato por pais y en el apendice el detalle por persona

Ojo aca solo se tienen que considerar las salidas que sean de esta semana y no se debe poner eso del riesgo

https://i.imx.to/i/2026/11/09/78gx91.jpg

https://i.imx.to/i/2026/11/09/78gx8y.jpg

than should be outside the parentheses.

In this preprint, Beyers et al. use ligands of both HaloTag and streptavidin to control protein–protein interactions. The approach is interesting, but at least two points need attention. 1. The evidence for Golgi-to-ER recycling of ManII is over-interpreted. The KDEL receptor is present throughout the Golgi and can retrieve KDEL-bearing proteins even from the trans-most cisternae (https://doi.org/10.1083/jcb.129.2.309). The return of ManII to the ER observed with BOLT may therefore reflect KDEL receptor–mediated retrieval of the KDEL-bearing complex, rather than the protein’s own recycling pathway. 2. Inducible and reversible control of the RUSH system has already been demonstrated The Introduction states that streptavidin–SBP binding is “virtually constitutive, prohibiting inducible interaction”, and this framing underpins the claim of novelty. Indeed, artificial ligands of streptavidin (ALiS) were developed specifically to make RUSH reversible and inducible (https://doi.org/10.1021/jacs.5b05672) and (https://doi.org/10.1002/cbic.201600640). ALiS allow bidirectional control of ManII transport, both ER-to-Golgi and Golgi-to-ER. These studies should be cited, and the novelty claims revised accordingly.

Niccolo Machiavelli’s The Prince explains how rulers can gain and maintain power. He believes leaders should be smart, prepared, and willing to make difficult decisions to protect their position. Machiavelli also argues that it is better for a ruler to be feared than loved, as long as people do not hate them. Overall, the book focuses on leadership, power, and doing what is necessary to keep control.

Robert P. Harrison’s article explains why Niccolo Machiavelli’s The Prince is still important today even though it was written over 500 years ago. Machiavelli believed that successful leaders needed to be realistic, clever, and willing to do whatever was necessary to maintain power. He argued that it was better for a ruler to be feared than loved because people can be unpredictable and often act in their own self-interest. The article uses Cesare Borgia as an example of a powerful leader who used strategy and control but was eventually defeated by unexpected events. Harrison explains that power and planning cannot control everything in life. His main point is that imagination and the ability to look beyond reality can sometimes create greater change than realism alone.

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It is interesting that they lack empathy and they manipulate others to get what they want

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Reply to the reviewers

We thank the reviewers for your thoughtful assessment of our study and for recognizing its technical advance. We appreciate the reviewers’ comments and suggestions, all of which we have carefully addressed in the revised manuscript.

Reviewer #1

1. In the first section of results, a self-cleaving 2A peptide is inserted upstream of the selection marker to improve selection of correctly tagged genes, as reported in the cited literature. To demonstate this in their PCR-tagging method, the authors tag the HNRNPA1 gene with the mNG-2A-NeoR strategy and find a tagging efficiency of 70% and more, in contrast with the 32% efficiency found with the original method developed by Fueller et al. (2020) (page 5). In support of this conclusion, only a representative image of cells showing mNG and HOECHST signal is presented, with no quantification (Fig1b). As this is a central result of the manuscript that allowed the authors to maintain the 2A-NeoR selection strategy for further investigation of sequence-level data, it is advisable to perform a more thorough quantification of the tagging efficiency by counting (with the help of image analyis software such as Fiji) the mNG+ cells and the total number of cells (HOECHST+), and calculate the % of mNG+ cells across different biological replicates (ideally, cells transfected in different days). A direct comparison (statistical analysis) with the % of mNG+ cells obtained by the original methodology (Fueller et al) would be desirable. In this way, the robustness of the improved tagging efficiency with the optimized method could be also evaluated within one single gene.

Re____sponses

• We have now quantified the fraction of mNG+ cells across three biological replicates for three different genes, using cells transfected in independent experiments. The representative images and quantitative analysis have been added to the revised Figure 1b.
• We also revised the corresponding text in the revised manuscript on page 5 (lines 143-151) to avoid overinterpreting the comparison with the tagging efficiency previously reported by Fueller et al. (2020). The previous experiments were performed in parental HEK293T cells using Zeocin-based selection, whereas the present experiments were performed in HEK293T NeoR-KO cells and G418 selection. Since several experimental parameters were not fully matched, we agree that a direct statistical comparison would not be appropriate. The revised text is:” Using the 2A-linked selection cassette, we tagged HNRNPA1, DDX21, and SLC16A1 in HEK293T NeoR-KO cells (HEK293T cells in which the neomycin-resistance (NeoR) gene linked to the T-antigen has been inactivated), with three independent biological replicates for each gene. … (Figure 1b). Quantification across biological replicates confirmed the reproducibility of the 2A-linked tagging workflow and support its use in subsequent characterization and optimization.”
• In addition, we changed the title of this Results section to “2A-linked selection and characterization of integration outcomes of CRISPR-Cas12a-assisted PCR tagging” to avoid the claim of direct improvement relative to the previous method. Nevertheless, the high and reproducible tagging efficiencies observed here support the use of the 2A-linked selection strategy for subsequent experiments.

  1. In the following line (page 5), authors talk about "correctly localized" signals, although no clear criterium of "correct" signal localization is explained, nor it is indicated anywhere in the example image. While in the following section(s) it is mentioned that diffuse signal may come from unspecific mNG expression of concatenated PCR cassettes, it would be still important to quantify the "correct" vs "uncorrect" mNG signal (i.e: nuclear vs cytoplasmatic) via image analysis.

__Re____sponse: __Thank you for pointing out the lack of localization definitions. In the revised Figure 1b, examples of diffuse cytoplasmic fluorescence are indicated by white arrows, and the fractions of the two localization categories (“correct” vs “uncorrect”) are quantified. Accordingly, the Results text was revised as “Following G418 selection, fluorescence microscopy showed that most mNeonGreen-positive cells displayed signals consistent with the expected gene-specific subcellular localization, whereas only a small fraction exhibited diffuse mNeonGreen fluorescence (Figure 1b)” on page 5 (lines 146-149). And the criteria used for classification are now described in the Methods and figure legend.

1. Of particular significance would be to clarify whether there is a relationship between the % of concatemer product and the presence of diffuse cytoplasmic signal. In several instances, the authors mention their previous finding that diffuse cytoplasmic signal usually results from unspecific expression of the fluorescent reporter within a concatenated PCR cassette. However, from Fig1b there is no evident correlation between % of concatemer reads and % of diffuse cytoplasmic signal. Moreover, Fig2c is cited but no arrows are indicated in the figure in contrast with what mentioned in the text (page 10). Moreover, it is unclear what the authors would like to highlight in the following paragraph when talking about the percentage of diffuse signal (the discrepancy of the concatemers vs diffuse signal? Please make the paragraph more clear). Overall, I think that showing actual microscopy data and image quantification of diffuse vs localized signal would be ideal instead of only mentioning the result in the text. Moreover, a clearer and more detailed discussion about the authors' interpretation of the discrepancy observed between the % of concatemers and % of diffuse cytoplasmic signal is needed, and how it relates to previous findings by Fueller et al. (2020).

Re____sponses

• We apologize for the confusion. In the revised Figure 1b we included the example of diffuse signals and also quantified the fraction of cells with diffuse signal across three biological replicates. We also show example of diffuse signals in revised Fig. S2d. In addition, we revised the paragraphs on page 22 (lines 494-514) to make the description of concatemers and diffuse signals clearer: “Since these reads represent donor-donor junctions rather than intact alleles, their abundance cannot be interpreted directly as concatemer copy number or as the fraction of cells harboring concatemeric integrations.
• In our previous study (Fueller et al., 2020), we proposed that concatemer formation may contribute to diffuse cytoplasmic fluorescence (examples indicated by white arrows in Figure 1b and Figure S2d) through unintended expression of mNG from downstream cassette copies. Although an SV40 PolyA terminator sequence was included downstream of NeoR, the U6 Pol III promoter driving crRNA expression has been reported to mediate Pol II-driven transcription that can contribute to the expression of downstream mNG sequences (Gao et al., 2018; Rumi et al., 2006; Figure 4a). We therefore quantified the fraction of cells displaying diffuse signal at eight loci under control conditions with unmodified donors and compared with the corresponding fractions of concatemer reads, to examine the relationship between concatemer formation and diffuse fluorescence.
• We found that the frequency of diffuse signal varied substantially across loci and did not closely track the fraction of concatemer reads. No diffuse signal was observed at the CLTC, DDX21, and HNRNPA1 loci. Only a few cells (

  1. It is expectable that the efficiency of PCR-tagging might vary if different preparations of the PCR products are used. The authors should add technical details in the method section that are currently missing regarding kits and protocols used for PCR, PCR purification/extraction, verification of PCR by sequencing (within the "Generation of plasmids and PCR cassettes"paragraph). This includes kits, polymerase type (Q5? Phusion?), extraction protocols (was the PCR gel-extracted?), sequence verification (was the extracted band sent for sequencing prior to perform PCR-tagging?) and so on. In particular, sequence verification by Sanger sequencing would help understand whether some small nucleotide changes were generated by PCR prior to cell transfection and should be added to the discussion part as source of possible mutations.

Responses

• The PCR cassettes were amplified using Velocity DNA polymerase (Bioline), following our previously published protocol (Fueller et al., 2020). As described there, other commercia high-fidelity polymerases like Phusion (Thermo Fisher Scientific), as well as self-purified Pfu-Sso7d DNA polymerase, can also be used.
• Sanger sequencing of PCR cassettes before transfection was not performed, as the cassette sequence is defined by the known template plasmid and the primer sequences. Template plasmid was verified by sanger sequencing and the primers were synthesized and purified by cartridge to ensure high fidelity/purity. Although small nucleotide changes introduced by PCR cannot be excluded as a potential source of mutations, but their overall contribution is expected to be minor given the high-fidelity nature of the polymerases.
• We have now added the experimental details to the Methods on page 33 (lines 817-827):” The PCR cassettes were amplified from either pMaCTag-2A-N07 (mNeonGreen) or pMaCTag-2A-N11 (mScarlet-i) template plasmid based on our previously published protocol (Fueller et al., 2020). In brief, PCR cassettes were amplified using a Velocity polymerase (Bioline) in a reaction mixture containing self-made HiFi buffer (200 mM Tris-HCl pH 8.8, 100 mM (NH₄)₂SO₄, 500 mM KCl, 1% [vol/vol] Triton X-100, 1 mg/ml BSA, 20 mM MgCl₂), 10 mM dNTPs (Bioline), 5 M betaine (Sigma-Aldrich), 200 ng template plasmid, and 10 µM each of M1 and M2 tagging oligos. The following cycling conditions were used: 3 min at 95°C; 30 cycles of 20 s at 95°C, 30 s at 64°C, and 45 s per kb at 72°C; 5 min at 72°C; and 4°C hold. After PCR, 0.4 µl DpnI was added and the reaction was incubated for 1 h at 37°C to digest the plasmid template. PCR products were verified by agarose gel electrophoresis to confirm product size. As our PCR reactions consistently yielded a single specific band of the expected size, column-based purification (Macherey-Nagel, NucleoSpin Gel and PCR Clean-up) was performed without gel extraction.”

  1. [OPTIONAL] A significant improvement of the study in terms of reproducibility would be to analyze, via microscopy image analysis (NOT by sequencing), the % of tagging efficiency as suggested above (see point 1 under the microscopy section) for the same gene (i.e. HNRNPA1 or other) with biological replicates consisting of cells transfected with PCR reactions prepared in different days, or [OPTIONAL] even analyzing how the tagging efficiency varies with PCR reactions purified with different kits or protocols (example: gel extraction vs PCR elution). Image analysis should include signal quantification with estimation of the % of tagging efficiency and statistical analysis. These experiment, not particularly demanding in terms of reagents and time (~3 weeks in total due to puromycin selection according to the workflow in Fig2b) would greatly help the reader understanding the robustness of tagging efficiency within single genes.

Re____sponses

• As described in our response to the comment 1, we have now quantified tagging efficiency by microscopy for three different genes across three independent biological replicates. For each replicate, cells were transfected with independently prepared PCR products (generated on different days, in some cases by different lab members). Fraction of mNG+ cells was quantified using Fiji, and the statistical analysis has been added to the revised Fig. 1b.
• We agree that systematically comparing different PCR purification methods could provide additional technical information. However, our PCR reactions consistently yielded one specific band of the expected size, we therefore performed a simple column-based purification to minimize the processing time. We have nevertheless clarified the experimental procedures in the Methods on page 34 (lines 824-827): “PCR products were verified by agarose gel electrophoresis to confirm product size. As our PCR reactions consistently yielded a single specific band of the expected size, column-based purification (Macherey-Nagel, NucleoSpin Gel and PCR Clean-up) was performed without gel extraction. All the primers and oligos used for cloning, validation and PCR cassettes are listed in Table S1.”

  1. The concentrations of pharmacological inhibitors used (stated in the result section at page 8) should be included in the method section (in the "Transfection" section) and in FigS2, along with details of their use in cell culture. "If NHEJ inhibitors were applied, cell culture media containing small molecules (KU0060648, NU7441, VE-822, M3814) were added after 8 hours of transfection and cells were incubated for 16 hours. Two days after transfection, cells were selected with 500 μg/ml neomycin (Sigma-Aldrich)". Was the drug kept in the medium during neomycin selection?

Response: We have revised Fig. S2a to highlight the selected concentrations of inhibitors in red on x-axis. We have also added the concentrations to the Methods section and revised the text accordingly on page 34 (lines 834-837): “Briefly, cells at ~70% confluency in one well of a 24-well plate were transfected with 100 ng of Cas12a helper plasmid (either impLbCas12a (Addgene #137715) or impLbCas12a-hGEM) and 900 ng PCR cassette. After 8 h, the transfection medium was replaced with either fresh culture medium. Where indicated, NHEJ inhibitors (200nM KU0060648, 1μM NU7441, 1μM VE-822, and 500nM M3814) were added at this time, and cells were incubated for an additional 16 hours before the inhibitor-containing media was replaced with fresh culture medium. One day after transfection, cells were expanded to a 6-well plate. Two days after transfection, cells were selected with 500 µg/ml G418 (Sigma-Aldrich).”

1. While the authors mention the absence of technical/biological replication within single genes, some graphs are presented with error bars (Fig1F, Fig2d, FigS2b) although no details are given in the text. What replication has been done? What do the error bars represent? What is the sample size?

Re____sponse: Thank you for pointing out this ambiguity. For the samples shown in the original Fig. 1f and Fig. 2d, sequencing libraries were prepared independently in different batches from the same population of tagged cells (from the same transfection), and were sequenced on separate batches. We initially displayed the variation between these technical measurements as error bars. But since they do not represent independent biological replicates, we agree that the error bars could be misleading, so we now show the results from a single sequencing batch for each condition (revised Fig. 1f and revised Fig. 2e). For Fig. S2b, the results of 8h siRNA knockdown were generated from two biological replicate, we have also clarified this point in the revised figure legends.

1. (pages 9-10, Result section): "We quantified the ratio of the HDR band intensity to the total intensity of both bands, defining this as "HDR purity". At the HNRNPA1 locus, we observed that both inhibition of c-NHEJ and synchronization of Cas12a increased HDR purity (Figure 3a)." How were the bands quantified to obtain the reported HDR purity %? Which software was used for band intensity quantification? Similarly, the information about how HDR purity was calculated with the anchor-seq data is lacking. These pieces of information are not present in the Method section and should be added.

Response: This information has been included in the Method section on page 36 (lines 903-905): ” The PCR products were analyzed by gel electrophoresis on 1% agarose/TAE, where the upper band corresponds to junctions generated by integration via c-NHEJ and the lower via HR. The intensity of both bands was quantified using Fiji (ImageJ 2.14.0/1.54f). HDR purity was quantified as the ratio of the HDR band intensity to the total intensity of both bands.” and on page 38 (line 966): ” In addition, HDR purity was calculated as the ratio of the HDR / (HDR + Indel) reads. All custom scripts were integrated into a Snakemake workflow, allowing automated execution of the analysis pipeline.”

Minor comments:

1. A few typos are present throughout the text. Examples: "because the probability of perfect insertion at both ends of the reporter are very low" (page 2); "With this 'mammalian PCR tagging' method constitutes a" (page 3); "In this work we address these gaps and employ selection markers fused in-frame to the tag of choice (e.g. mNeonGreen) using a 2A peptide spacing the two protein domains ensures that" (page 4); "but increased the off-target frequency for TOMM20 by 0.6-fold? (page 9); "from the wide-type and tagged cells" (page 17); "Together, our improvements outline an robust strategy" (abstract). Some other might be present, please check carefully.

Re____sponse: We apologize for the typos. These have been fixed in the revised manuscript. We also checked to make sure other typos are corrected.

1. The Acknowledgement part seems incomplete, and the author contributions are not listed.

Re____sponse: The Acknowledgement part has been revised and author contributions have been added.

1. Can the authors explain the concept/meaning of "index hopping" in the text (page 6)?

Response: We have revised the text on page 6 (lines 207-213): “In Tn5-Anchor-Seq, the customized barcode strategy enables the pooling of a large number of samples immediately after Tn5 tagmentation, but this pre-PCR pooling can introduce index hopping — the misassignment of reads to an incorrect sample barcode through cross-amplification. To assess the extent of index hopping from pre-PCR pooling, we designed a dedicated experiment in which reads assigned to barcode-gene combinations not expected in the pool were counted as misassigned, and their fraction relative to total reads was calculated as the index hopping rate. The observed rates ranged from 0.05% to 6%.”

1. The FigS2g does not exist (cited at page 8). Did the authors mean f?

Re____sponse: We apologize for the incorrect figure citation. It has been corrected in the revised manuscript.

1. There is a discrepancy between Figures and labels regarding FigS3 at page 10: "The PS&Btn modifications on PCR cassettes reduced the frequency of concatemeric reads by 0.5- to 1.7-fold for most genes, while no effect was seen for CANX and SLC16A1 (Figure 4c and Figure S3d). As expected, inhibition of NHEJ with KU, NU, or M3814 decreased the concatemeric integration, while treatment with VE or the use of Cas12a-hGEM increased concatemer frequency at specific loci, such as CANX, DDX21, and SLC16A1 (Figure 4d, 4e, and Figure S3e). Inhibition of MMEJ by knocking down Polθ reduced concatemeric integration at the EMD and SLC16A1 loci but not at the LMNA locus (Figure S3e)." FigS3e does not exist, instead S3d seems to contain what S3e was supposed to show.

Re____sponse: We apologize for these mistakes. We have carefully reviewed the entire manuscript to ensure that all Figures and labels are consistent.

1. Figure 5b is never cited in the main text, nor elsewhere in the manuscript.

Re____sponse: We apologize for the typo. Fig 5b was mistakenly referred to as Fig 5a. This has now been fixed.

1. In the FigS2c caption, remove the sentence "All PCR cassettes used in this experiment were modified with PS and Btn." since both modified and unmodified PCR cassettes are shown in the panel.

Re____sponse: We apologize for this mistake. The Fig. S2c caption has been revised.

1. The scale bar in the microscopy images (Fig1b, Fig5c) is not clearly visible, please fix the thickness.

Re____sponse: Thanks for pointing this out. We have increased the thickness of the scale bars in the revised Figure 1b and Figure 5c.

1. In Fig5d, some lanes are highlighted in red although no explanation is given in the figure caption or in the text of what red color lanes represent.

Re____sponse: The red lanes indicate the clones that were subjected for sequencing. The Fig. 5d caption has been revised accordingly.

1. In FigS1, different graphs of different panels have the same legend, that is placed only within panel f. The same applies for FigS3. It would help readability to state where to find the legend in the figure caption or to rearrange the panels so that the legend appears next to each graph.

Re____sponse: Thank you for pointing this out. The plots in Fig. S1b, d, and f use the same symbols and colors for the integration outcome categories, with a shared legend next to panel f. We have clarified this in the revised Fig. S1b legend as well as the caption to improve the readability. We also make sure the other figure legends are clear in the revised manuscript.

11.Throughout the schematics used to exemplify the method workflow or cassette architecture there are several abbreviations that are not expanded in the relative figure caption. Examples: Figure 1a (HA, mNG, NeoR, U6, crRNA), Figure 1b (ONT, BC, Tn5) [...]. While some of them can be evinced in the main text, expanding each abbreviation in the respective figure caption can improve the figure readability.

Re____sponse: We have included the abbreviations in the respective figure captions throughout the revised manuscript.

1. In Fig1e, several symbols are present in the IGV snapshots whose meaning can only be guessed (what are purple vertical lines? What do numbers and letters indicate and what do the color indicate?). This information should be added as legend in the panel or explained in the figure caption. Similarly, it is not clear what the IGV snapshot indicates, it would be useful to add vertical lines in the IGV snapshot indicating, for example, the 5'-HA/3'-HA portion or something else that could help the reader understand what portion of the cassette is indicated.

Re____sponse: Thank you for the suggestions. We have added vertical lines to the IGV snapshots in the revised Fig. 1e to indicate the approximate positions of 5’-HA junctions. We have also expanded the figure caption to explain the IGV annotations: “(e) Schematics illustrating genomic integration scenarios following PCR tagging and corresponding IGV snapshots showing sequencing-read alignments: i. HDR-mediated integration; ii. NHEJ-mediated deletions and insertions; iii. Off-target integration; iv. Concatemeric integration. Black dashed lines connect each schematic to the genomic interval displayed in the corresponding IGV snapshot. In the IGV snapshots, grey bars represent aligned sequencing reads, whereas mismatches to the reference sequence are shown as colored vertical marks according to the standard IGV nucleotide color scheme (A, green; C, blue; G, brown; T, red). Purple vertical marks denote insertions, with numbers indicating the base-pair length of the insertion. Gaps or thin connecting lines within individual reads indicate deletions, with numbers indicating the size of deletion. Blue vertical dashed lines indicate the approximate positions of the 5’-HA junctions.”

1. Please specify that FACS was used only for the seamless tagging experiment, as it's unclear in the text. Were sorted cells seeded as one cell per well, or were they collected in one single tube and then separated? Why is Fig5b showing the sorting of mNG fluorescent cells while the result and method only mScarlet-i is mentioned (possibly wrong figure)?

Re____sponses

• FACS was only used for the seamless tagging experiment, and single cells were seeded as one cell per well. We have added this information to the Methods text on page 35 (lines 877-884): “During seamless PCR tagging, following G418 selection, cells were harvested, washed with PBS…… Single mScarlet-i-positive cells were sorted directly into separate wells of 96-well plates containing……”
• We have also revised Fig. 5b, in which the mScarlet-i was used as the fluorescent protein.

  1. In FigS2, panels e-f show the same variable (fraction of HDR reads). For EMD locus, however, there is a possible discrepancy on the fraction shown in e (yellow bar, PS&Btn) and f (grey bar, no treatment, modified oligos). Can the authors explain this apparent discrepancy?

Re____sponse: Thank you for pointing this out. Panels e and f (correspond to the revised Fig. 2d and S2e) were generated from different batches of transfection. The difference of EMD values is attributed to batch-to-batch transfection variability. Therefore, comparisons within each panel were performed within the same transfection batch. We have clarified this in Methods text on page 34 (lines 844-847): “Independent experiments shown in separate figure panels were performed in distinct transfection batches unless otherwise indicated. Because absolute editing efficiencies can vary between transfections, treatment effects were evaluated relative to the corresponding controls within each experimental batch. ”

1. Looking at some figures, such as Fig2, it is sometimes hard to relate the results to a "bigger picture". For example, Fig2d depicts "% of HDR integrations", while Fig2f shows "Fraction of HDR reads (%)". Besides possible confusion regarding the name of the variable analyzed, the caption of Fig2f explains that the relative panel contains the data in Fig2d. As Fig2d contains % of HDR-mediated integration of unmodified cassettes ranging 60-85%, one would expect to find the same percentages in Fig2f, under unmodified, control cassette; however, on average the fraction of HDR reads in these samples is only 10-20%. How is this possible? Is Fig2f including unmapped reads or reads mapped to off-target loci?

Re____sponse: We apologize for the confusion. We agree that the original description was not reader-friendly. We have restructured Results section 2 (pages 7-9). We now first describe the full spectrum of tagging outcomes across all gene loci and treatment conditions using the metrics “fraction of tagging outcomes”. Then we present in detail the “HDR efficiency” across conditions. In addition, we have clarified these metrics in Methods on page 37 (lines 961-966):” The total read count for tagging outcomes was defined as HDR + Indel + off-target + concatemer reads, whereas genomic integrations (Figure 1c) were defined as HDR + Indel + off-target reads, excluding concatemer reads. Thus, outcome fractions in the holistic analysis (Figure 2d) were normalized to all classified tagging outcomes, while event efficiencies/frequency (HDR, Indel, off-target) were calculated relative to genomic integrations only.”

1. The graph in Fig3d displays Log2FoldChange of indel frequencies. To have a better understanding of the data, it is advisable to show an additional graph with "raw" indel frequencies for each gene, as side-by-side bars (similar to the graph in Fig2c). The same applies for the fold change shown in Fig3g.

Re____sponse: Thank you for this suggestion. We have revised the figure to include the additional graphs showing the raw frequencies under the control conditions (revised Fig. 3d, 3g), and moved the Log2FoldChange graph to revised Fig. S3c, S3e. We have also included the side-by-side bars showing the raw frequencies under all conditions in the revised Fig. S3b, S3e.

1. [SUGGESTION TO IMPROVE CLARITY] Relative to the comments above (15-16), it would be advisable to add one or more graphs that summarize the read composition, similar to what has been done for Fig1f, for all genes analyzed in the different conditions. For example, it would be advisable to add a supplementary figure with one graph per gene analyzed (8 graphs in total) in the form of the one displayed in Fig2f, with stacked bars instead of boxplots as shown in Fig1f. This will help the reader understanding better and compare side-by-side the effect of the treatments applied in read composition for each gene.

Re____sponse: Thank you for this suggestion. We agree that a holistic overview of the outcome composition is important and helpful to illustrate the overall effects of different conditions. We have therefore showed the full spectrum of tagging outcomes across all gene loci and treatment conditions in the revised Fig. 2d and S2e. We have also revised the results text accordingly on pages 12-13 (lines 333-355).

Reviewer #____2

1. The most important shortcoming of this manuscript is the lack of biological replicates. The authors present results from multiple loci, but in this setting, variation will be largely driven by locus-specific effects rather than true experimental variation, which can be substantial for these types of methods. This makes it difficult for readers and potential users to evaluate how robust this pipeline is. The authors must present biological replicates for at least a small number of loci with their finalized method.

o Significant time and resources will be needed.

Re____sponse: Thank you for this valuable suggestion. We agree that biological replicates are critical for demonstrating the reproducibility and robustness of our workflow. We have therefore revised Fig. 1b, to include quantification of the fraction of mNG+ cells across three biological replicates for three different genes. These replicates were obtained from independent transfection experiments and were subjected to statistical analysis. The text on page 5 (lines 143-151) has also been revised accordingly.

1. The authors characterize clonal genotypes following seamless PCR tagging at two loci. They report that 2/6 isolated clones of HNRNPA1 have the desired edit, while 3/3 clones of SLC16A1 have the desired edit. However, the small number of amplicons sequenced makes it difficult to draw robust conclusions about clonal genotype outcomes. The authors should perform additional amplicon sequencing to strengthen these conclusions. In addition, they should sequence the incorrectly sized products generated from the junction-PCRs to gain further insight into alternative genotypes and incorrect editing that may occur with this method.

o Additional sequencing can easily be accomplished if cell lines are still available. An estimated time of about 1-2 weeks for cell thawing, genome extraction, amplicon sequencing, PCR purification, and outsourced Plasmidsaurus sequencing.

Re____sponse: Thank you for this important comment. We agree that sequencing more clones would robustly determine the frequencies of different genomic integration outcomes following seamless PCR tagging. However, our intention was not to quantitatively mapping full spectrum of editing outcomes, rather demonstrate that the two-cassette strategy can generate seamless integrations at different gene loci. We have therefore revised the Results text on pages 26 (lines 603-614) to avoid overclaiming regarding the genotype frequencies.In addition, we have revised the corresponding Discussion text on page 30 (lines 704-713) accordingly.

1. It is not obvious how the authors were able to accurately quantify concatemeric integrations with the Tn5-anchor-seq tool. Specifically, the schematic in Figure 4a makes it clear that concatemers have multiple binding sites for the PCR primer. If these binding sites give rise to multiple products from the same template, the quantification would be inaccurate. The authors should comment on their considerations in suppressing the formation of secondary products, such as the use of a non-strand displacing polymerase, and in ensuring that their quantifications are accurate, including any bioinformatic steps.

o The authors can clarify methods and adjust results presented in the manuscript in 1-2 days.

Re____sponse: Thank you for pointing this out. The scenario where a single template generates multiple PCR products would require amplification of an intact concatemer. However, in our Tn5-anchor-seq, Tn5 tagmentation precedes PCR so that concatemers are fragmented before PCR. Primer sites from a N-copy concatemer therefore reside on separate tagmented fragments, which are amplified independently. So, the concatemer-derived read counts scale with the amount of concatemeric sequence present in the library. Potential secondary products are further minimized by the nested two-round PCR design, biotin-streptavidin enrichment of cassette-primed amplicons, limited first-round PCR (15 cycles), hot-start polymerase, and size selection of products > 500bp. In bioinformatics analysis, each read is counted once after demultiplexing, mapping, filtering and removal of secondary and supplementary alignments. Importantly, this metric reports the fraction of concatemeric sequence, rather than the number of concatemeric integration events per allele. We have clarified the interpretation of concatemer reads in the revised manuscript on page 22 (lines 494-497).

1. The authors begin the results section by describing how their revised tagging cassette improves selection efficiency. It appears that they compare their newer data to data that were obtained for their previous manuscript, which were likely obtained at different points in time, potentially by different individuals, and using different sets of reagents, which makes direct comparisons impossible. It would be beneficial to include at least one side-by-side experiment to compare the authors' new method with their previous method to conclusively demonstrate any improvement.

Re____sponses

• We agree that our original text overinterpreted the comparison with the tagging efficiency previously reported by Fueller et al. (2020). The previous experiments were performed in parental HEK293T cells using Zeocin-based selection, whereas the present experiments were performed in HEK293T NeoR-KO cells using G418 selection. Since several experimental parameters were not fully matched, we recognize that a direct quantitative comparison is not be appropriate.
• We have therefore removed the sentence “compared to 32% when employing a cassette in which the selection marker was inserted as a separate gene (Fueller et al., 2020). ” in the revised Results text on page 5. Instead, we now report the quantification across biological replicates to show the reproducibility of the workflow using the new 2A-linked selection cassette, thereby supporting the use of the 2A-linked selection strategy for subsequent experiments.
• In addition, we changed the title of this Results section to “2A-linked selection and characterization of integration outcomes of CRISPR-Cas12a-assisted PCR tagging” to avoid the claim of direct improvement relative to the previous method.

  1. Data and method presentation: For much of the manuscript, the authors provide detailed descriptions of experiments and methods. However, the authors should provide more detail in the figure legends about the concentration and timing of chemical inhibitor treatments.

Re____sponse: Thank you for pointing this out. We have added the concentrations and treatment details to the Methods section and revised the text on page 34 (lines 834-837).

6.Replication and statistical analysis: While the authors mention their lack of replicates, and reason multiple loci and conditions compensate for this, replication of their experiments is still necessary in order to draw meaningful conclusions of their data (see comment above).

Re____sponse: As described in the response to the first comment, we have revised Fig. 1b, to include quantification of the fraction of mNG+ cells across three biological replicates for three different genes. The text on page 5 has also been revised accordingly.

Minor Comments:

1. The authors clearly identify and cite the prior work on which their method is based.

Re____sponse: We thank you for this kind comment.

1. The discussion focuses on the findings and limitations of the authors' specific study. Readers would benefit from a conceptual comparison of the strengths and weakness of their method to other similar technologies in this space, which would allow researchers to make better informed decisions about the appropriateness of this method for their own work.

Re____sponse: Thank you for this suggestion. We have added a paragraph in Discussion on page 31 (lines 718-727) to compare with similar technologies.

1. The text and line of the scale bar on all microscopy images needs to be enlarged for readability.

Re____sponse: All the scale bars of the microscopy images have been enlarged in the revised manuscript.

1. Text has inappropriate question mark in "... off-target frequency for TOMM20 by 0.6 fold?".

Re____sponse: The question mark has been removed.

1. Fold-changes in the text are described in an unconventional and unintuitive manner. For example, the authors note a 0.2 to 0.7-fold reduction in off target frequency contrasted against a 0.6-fold increase, which appear to be two different interpretations of fold-changes. These results might be more accurately represented by using a 1-fold change as the baseline for no change. Alternatively, the authors could provide this measure as percent decreases and increases.

Re____sponse: Thank you for pointing this out. We have revised the text to express decreases and increases as percent changes relative to the control condition, while retaining log2 fold-change values in the heatmaps. The revised text show on lines 436, 447, 448, and 517.

1. The authors mention that an example of diffuse cytoplasmic fluorescent signals observed in microscopy can be seen in Figure 2c, which are labeled with white arrows. A figure with this description is not present in the manuscript.

Re____sponse: We apologize for the confusion. In the revised Fig. 1b and S2d, examples of diffuse cytoplasmic fluorescence are indicated by white arrows. Accordingly, the Results text was revised on page 22 (lines 498-500).

1. Schematics in Figure 5 show mNeonGreen, however, Figure 5c is labeled as mScarlet. The Sauthors should clarify which fluorescence tag was used.

Re____sponse: We have revised Fig. 5a by replacing “mNG” with the generic word “tag”, as this panel is a general schematic to compare the two protein-tagging strategies. We also corrected Fig. 5b and 5d to indicate mScarlet-i, which is the fluorescent tag used in this part.

1. Figure S3e is mentioned in the text, however, the supplemental material does not contain a 3e.

Re____sponse: The typo has been corrected in the revised manuscript.

1. The authors have developed a fantastic pipeline to quantify tagging outcomes holistically, and in Figure 1f they visualize the results of the pipeline in an intuitive and informative manner. However, for the remainder of the manuscript, they proceed not to use this visualization and instead focus on the effects of their experimental parameters on individual outcomes or a ratio of two outcomes ("HDR purity"). This choice makes it difficult to clearly evaluate the effects of the parameters on all outcomes, and the "HDR purity" metric, although useful to compare against the gel-based readout, is difficult to interpret. We would recommend including the holistic visualization from Figure 1f for all experiments, in the supplement if necessary, for example as a large grid that depicts the frequency of all outcomes for each experimental condition by locus. This will make it much easier for readers to evaluate how a given experimental parameter affects the frequency of different editing outcomes.

Re____sponse: Thank you for this important suggestion. We agree that a holistic overview of the outcome composition is important to illustrate the overall effects of different conditions. We have therefore restructured the Results section 2 by first showing the full spectrum of tagging outcomes across all gene loci and treatment conditions in the revised Fig. 2d and S2e. We then described the HDR efficiency in the revised Fig. 2e, 2f, and S2g. The corresponding text has also been revised accordingly (pages 12-13, lines 333-355). We have also revised the description about HDR purity results on page 17 (lines 414-423) to make it clearer.

1. While the authors detail their rationale in selecting loci to tag in a previous publication, it is important to include the information in the current manuscript as well.

__Re____sponse: __We have included this information in the revised manuscript on page 11 (lines 305-311).

1. The authors present many figures that look similar but display different quantities. To improve readability, they should use more descriptive figure titles and axis labels so that the main findings and quantities are immediately clear. This is particularly needed in Fig.2d, where the variable plotted is not clear.

Re____sponse: We have restructured Figure 2 to provide a more holistic overview of the full spectrum of tagging outcomes. The original Fig. 2d has been revised as Fig. 2e with the y-axis labeled “HDR efficiency” and the condition indicated as “Unmodified, no treatment” on top. We have also revised the axis labels and results description on page 12 (lines 349-355) for greater clarity.

1. Two aspects of the final method may (or may not) represent limitations that would be helpful for the authors to discuss. First, the authors use a Cas12a with expanded PAM range, which has the advantage of increasing the number of available cut sites but also has the potential to lead to more off-target cutting, which can cause secondary mutations in the selected cell populations. Second, tagging the 3′ UTR may lead to differences in transcript and protein stability (especially due to the remainder of the 2A site). It would be helpful for the authors to include their perspectives on these issues and if they took efforts to address them.

Re____sponses

• Thank you for raising these two important points. Regarding the expanded-PAM Cas12a variant, we have added this point explicitly to the limitations paragraph in Discussion on page 32 (lines 757-759): “The use of an expanded-PAM Cas12a variant may also alter off-target activity relative to wild-type Cas12a (Kleinstiver et al., 2019). Thus, comprehensive off-target profiling is needed in applications where genomic integrity is critical. ” Systematic off-target characterization was beyond the scope of the current study, but we consider this an important direction for future work, particularly for therapeutic applications.
• Regarding the 2A site remainder and 3’-UTR effects, we partially address the concerns about protein stability/integrity by tagging mNG-FKBP degron at four loci and confirmed efficient inducible depletion of the tagged protein following degron induction (revised Fig. S4). This demonstrates that the integrated fusion proteins are expressed and that the degron domain remains structurally accessible. However, we acknowledge that direct measurement of endogenous protein activity will be important in future studies. We have therefore added discussion of this point in limitations paragraph on page 32 (lines 760-764).

  Reviewer #____3

1. All optimization experiments are performed in HEK293T-N cells, which are aneuploid/polyploid, permissive for genome editing and have atypical DNA repair dynamics. While acceptable for method development, this limits generalizability, especially given the strong locus and pathway dependent effects observed. DNA repair pathway balance (HDR vs NHEJ vs MMEJ, etc.) can be cell-type specific. The improvements reported here may not generalize to non-transformed cells, stem cells, cancer cell lines, or other therapeutically relevant cell types. Selecting a small, representative gene set (2-3 genes) and performing controlled tagging experiments across a panel including cancer cell lines, non-transformed cells (like hTERT immortalized RPE-1 cells), and embryonic stem cells (H1 or H9) could provide readers with a clearer view of integration and tagging efficiencies across diverse cellular contexts. Additionally, assessing the effects of pathway modulation in these cell types would help determine how broadly generalizable these strategies are increasing the impact of the method detailed herein.

Re____sponses

• Thank you for raising this important point. We fully agree that cell-type generalizability is very important. HEK293T NeoR-KO cells were selected for systematic optimization because their high editing permissiveness enables robust detection of differences across conditions and loci, rather than because they are representative of the broader range of cell types.
• To assess the portability of our workflow, we tested it in two additional cell lines. First, we applied the optimized standard PCR tagging protocol — using PS- and Btn- modified donors in combination with 200 nM KU0060648 — to HeLa cells. Tagging of HNRNPA1, DDX21, and TOMM20 was evaluated by fluorescence microscopy and flow cytometry. The fraction of mNG+ cells at expected localization were comparable to the observations in HEK293T-N cells. Second, we applied the seamless tagging strategy to HepG2 cells at HNRNPA1 locus and confirmed the comparable tagging efficiency via fluorescence microscopy. All these data are now summarized in Supplementary Note 1.
• We note that sequencing-based integration outcome characterization was not performed in either HeLa or HepG2 cells. We have clarified this in the revised Discussion on page 32 (754-757).

  1. While the manuscript presents a clear and well-supported optimization of CRISPR-Cas12a assisted PCR tagging, it would benefit from a more explicit contextual comparison to other contemporary endogenous tagging approaches, such as Cas9-mediated HDR with plasmid donors. Ideally, a single or small panel of genes could be compared to assess the benefits and drawbacks of each technique. Even a conceptual discussion outlining relative strengths, limitations, and use cases would help readers better position the proposed method within the broader genome-editing landscape. Such a comparison would clarify when PCR tagging is the most appropriate choice and where alternative strategies may offer advantages, thereby enhancing the interpretability and practical impact of the study.

Re____sponse: Thank you for this suggestion. We have revised the Discussion to include a dedicate paragraph for comparison with other similar technology on page 31 (lines 718-727).

1. Lack of functional validation beyond localization. Localization is used as the primary functional readout. Including limited functional validation (e.g., protein abundance, stability, or activity) for a representative target would strengthen the claim that optimized conditions preserve protein function, since donor modifications and DNA repair pathway inhibition could affect protein dosage or integrity. Alternatively, explicitly acknowledging this limitation would better frame the scope of the functional conclusions. At a minimum this needs to be discussed carefully.

Re____sponses:

• Thank you for this important suggestion. We agree that subcellular localization alone provides limited evidence for functional protein integrity. Thus, we applied mNG-degron tagging at HNRNPA1, DDX21, CANX, and KIF11 loci to confirm protein integrity beyond localization. Following degron induction, efficient depletion of the mNG-tagged proteins was confirmed by microscopy, flow cytometry, and western blot. The combination of correct subcellular localization and successful inducible degradation indicates that the tagged fusion proteins are expressed and structurally intact at the endogenous locus.
• The results are presented in revised Fig. S4, and a brief description has been added to the Results section on page 23 (lines 530-539).
• We noted that these results are not full functional validation, the degradation efficiency does not directly show whether native protein activity is preserved. We have revised the Discussion on page 32 (lines 764-767) to acknowledge this limitation.

  1. The effects of donor cassette size and sequence composition on integration efficiency and concatemer formation are not systematically explored. Because larger or more complex payloads may engage repair pathways differently, a brief discussion of how donor size might influence the reported improvements would help contextualize the results. If extra curious, a single reliably editable target gene could be used to assess and explore the effect of varying payload composition.

Response: Thank you for this suggestion. The mNG-degron cassette described in the revised Fig. S4 and also our response to Major point 3 is larger and compositionally more complex, as it contains an additional degron sequence and an HA epitope tag. With this degree of payload size expansion, we didn’t observe any obvious reduction in performance with respect to PCR amplification or tagging efficiency assessed via microscopy. However, we acknowledge that the effects on HDR efficiency and concatemer formation were not evaluated specifically. We have now discussed this limitation in the revised manuscript on page 31 (lines 728-735).

Minor ____Comments:

1. The frequency of concatemeric integrations is inferred from junction-centric Tn5-Anchor-Seq data rather than direct measurement of intact alleles, which limits the ability to determine concatemer copy number, allelic structure, or the presence of complex rearrangements within a single genomic locus. While this limitation is acknowledged, it should be more clearly emphasized when interpreting the magnitude of concatemer suppression and improvements in genomic homogeneity, particularly given the polyploid nature of HEK cells. Clarifying that junction counts do not directly equate to allele counts would help prevent overinterpretation of these results and better frame the conclusions drawn from the sequencing data.

__Response____: __Thank you for this suggestion. We have revised the corresponding text on page 22 (lines 495-497) to avoid overinterpretation of the concatemer results.

1. Inhibition of DNA-PKcs emerges as one of the most effective strategies for increasing HDR-mediated integration and suppressing undesired byproducts in this study. However, the broader practicality of this approach is not explored in depth. DNA-PKcs inhibitors can introduce cytotoxicity, alter cell-cycle progression, or induce stress responses that may be incompatible with certain experimental systems or long-term phenotypic analyses. A more explicit discussion of these limitations, including guidance on dosing, duration of treatment, and scenarios in which donor modification alone may be preferable, would help readers/researcher assess the applicability of this strategy across different cell types and use cases.

__Response____: __We have expanded the discussion of DNA-PKcs inhibitors in the revised manuscript on page 29 (lines 667-670).

1. The study focuses exclusively on C terminal tagging, and the applicability of the optimized workflow to N terminal or internal tagging configurations is not examined. Because tagging position can substantially influence repair outcomes and expression, a brief discussion of whether the observed improvements are expected to generalize beyond C terminal insertions would help clarify the scope of the method.

__Response____: __We have expanded the limitation paragraph in Discussion on page 31 (lines 736-743).

1. Clarify the rationale for inhibitor concentrations in the main text, not only in supplement.

__Response____: __We have revised the text on page 11 (lines 295-297).

1. Define all abbreviations at first use (e.g., c-NHEJ, MMEJ) for clarity.

__Response____: __The abbreviations have been defined in the revised manuscript.

1. Explicitly state sample sizes for microscopy-based quantifications.

__Response____: __This has been addressed.

7.Clarify how index hopping was quantified and controlled in pooled sequencing analysis.

__Response____: __We have clarified this in the revised manuscript on page 6 (lines 207-213).

1. Briefly describe computational resource requirements for the analysis pipeline.

__Response____: __Thank you for this suggestion. We have included this information in the Methods text on page 38 (lines 971-975).

1. State whether the "seamless" tagging method was tested with donor modifications and inhibitors (Assumption is no from your drawings in Figure 5).

__Response____: __We apologize for the missing information. Seamless tagging was performed using PS&Btn-modified donor in combination with KU treatment, as this was the best-performing condition identified in the standard PCR-tagging experiments. We have now clarified this in revised Fig. 5b and Results text on page 26 (lines 593-595).

1. Clarify whether donor-end modifications affect PCR yield or fidelity. Also, discuss cost implications of biotinylated/modified primers for scalability

__Response____: __We did not observe any obvious reduction in PCR performance when using modified primers, although PCR yield and fidelity were not quantified as separate endpoints. We have therefore clarified this as well as the primer cost in Discussion on page 29 (lines 660-664).

1. State whether sequencing depth was normalized across conditions.

__Response____: __Thank you for pointing this out. Sequencing depth was not explicitly normalized across conditions as all reported metrics are per-sample fractions. However, we ensured that sequencing depth was sufficient for all samples, such that fraction estimates were not biased by low coverage. This is now stated in the Methods on page 38 (lines 966-969).

1. Abstract: replace 'an robust strategy' with 'a robust strategy'.

__Response____: __This has been fixed.

1. Results section: ensure consistent use of 'Cas12a-hGEM' vs 'hGEM-Cas12a'.

__Response____: __It’s now consistently “Cas12a-hGEM” in the revised manuscript.

1. Figure 3g: remove stray question mark after '0.6-fold?'.

__Response____: __It has been removed.

1. Methods section: standardize units (µM vs uM) throughout.

__Response____: __We now use µM throughout the revised manuscript.

1. Figure references: inconsistent capitalization of figure labels (Figure vs Fig.).

__Response____: __We now use consistently “Figure” in the revised manuscript.

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Referee #3

Evidence, reproducibility and clarity

Improved cloning-free one-step CRISPR-Cas12a-assisted tagging of mammalian genes using PCR generated reagents (PCR tagging)

Peer Review Summary:

This manuscript presents a substantive and technically rigorous refinement of the previously published CRISPR-Cas12a assisted PCR tagging method for mammalian cells. Building on the original framework, Lou et al. introduce donor cassette engineering, repair pathway modulation, and an advanced long-read sequencing-based analysis pipeline to systematically improve tagging fidelity and to quantitatively characterize integration outcomes. The work is carefully executed, clearly written, and demonstrates a strong understanding of both the strengths and limitations of PCR-based genome editing strategies. A major strength of the study is its emphasis on measurement rather than inference. The use of Tn5-Anchor-Seq coupled with nanopore long-read sequencing provides a detailed and largely unbiased view of on-target HDR events, NHEJ-mediated insertions, concatemer formations, and off-target integrations. This addresses a known weakness of fluorescence- or selection-based readouts, which can overestimate true "precise" editing outcomes. The authors are appropriately cautious in interpreting their data and explicitly acknowledge the residual heterogeneity that persists even under optimized conditions.

Major Points:

1. All optimization experiments are performed in HEK293T-N cells, which are aneuploid/polyploid, permissive for genome editing and have atypical DNA repair dynamics. While acceptable for method development, this limits generalizability, especially given the strong locus and pathway dependent effects observed. DNA repair pathway balance (HDR vs NHEJ vs MMEJ, etc.) can be cell-type specific. The improvements reported here may not generalize to non-transformed cells, stem cells, cancer cell lines, or other therapeutically relevant cell types. Selecting a small, representative gene set (2-3 genes) and performing controlled tagging experiments across a panel including cancer cell lines, non-transformed cells (like hTERT immortalized RPE-1 cells), and embryonic stem cells (H1 or H9) could provide readers with a clearer view of integration and tagging efficiencies across diverse cellular contexts. Additionally, assessing the effects of pathway modulation in these cell types would help determine how broadly generalizable these strategies are increasing the impact of the method detailed herein.
2. While the manuscript presents a clear and well-supported optimization of CRISPR-Cas12a assisted PCR tagging, it would benefit from a more explicit contextual comparison to other contemporary endogenous tagging approaches, such as Cas9-mediated HDR with plasmid donors. Ideally, a single or small panel of genes could be compared to assess the benefits and drawbacks of each technique. Even a conceptual discussion outlining relative strengths, limitations, and use cases would help readers better position the proposed method within the broader genome-editing landscape. Such a comparison would clarify when PCR tagging is the most appropriate choice and where alternative strategies may offer advantages, thereby enhancing the interpretability and practical impact of the study.
3. Lack of functional validation beyond localization. Localization is used as the primary functional readout. Including limited functional validation (e.g., protein abundance, stability, or activity) for a representative target would strengthen the claim that optimized conditions preserve protein function, since donor modifications and DNA repair pathway inhibition could affect protein dosage or integrity. Alternatively, explicitly acknowledging this limitation would better frame the scope of the functional conclusions. At a minimum this needs to be discussed carefully.
4. The effects of donor cassette size and sequence composition on integration efficiency and concatemer formation are not systematically explored. Because larger or more complex payloads may engage repair pathways differently, a brief discussion of how donor size might influence the reported improvements would help contextualize the results. If extra curious, a single reliably editable target gene could be used to assess and explore the effect of varying payload composition.

Minor Points:

1. The frequency of concatemeric integrations is inferred from junction-centric Tn5-Anchor-Seq data rather than direct measurement of intact alleles, which limits the ability to determine concatemer copy number, allelic structure, or the presence of complex rearrangements within a single genomic locus. While this limitation is acknowledged, it should be more clearly emphasized when interpreting the magnitude of concatemer suppression and improvements in genomic homogeneity, particularly given the polyploid nature of HEK cells. Clarifying that junction counts do not directly equate to allele counts would help prevent overinterpretation of these results and better frame the conclusions drawn from the sequencing data.
2. Inhibition of DNA-PKcs emerges as one of the most effective strategies for increasing HDR-mediated integration and suppressing undesired byproducts in this study. However, the broader practicality of this approach is not explored in depth. DNA-PKcs inhibitors can introduce cytotoxicity, alter cell-cycle progression, or induce stress responses that may be incompatible with certain experimental systems or long-term phenotypic analyses. A more explicit discussion of these limitations, including guidance on dosing, duration of treatment, and scenarios in which donor modification alone may be preferable, would help readers/researcher assess the applicability of this strategy across different cell types and use cases.
3. The study focuses exclusively on C terminal tagging, and the applicability of the optimized workflow to N terminal or internal tagging configurations is not examined. Because tagging position can substantially influence repair outcomes and expression, a brief discussion of whether the observed improvements are expected to generalize beyond C terminal insertions would help clarify the scope of the method.
4. Clarify the rationale for inhibitor concentrations in the main text, not only in supplement.
5. Define all abbreviations at first use (e.g., c-NHEJ, MMEJ) for clarity.
6. Explicitly state sample sizes for microscopy-based quantifications.
7. Clarify how index hopping was quantified and controlled in pooled sequencing analysis.
8. Briefly describe computational resource requirements for the analysis pipeline.
9. State whether the "seamless" tagging method was tested with donor modifications and inhibitors (Assumption is no from your drawings in Figure 5).
10. Clarify whether donor-end modifications affect PCR yield or fidelity. Also, discuss cost implications of biotinylated/modified primers for scalability
11. State whether sequencing depth was normalized across conditions.

Spelling/Grammar/Other Errors:

1. Abstract: replace 'an robust strategy' with 'a robust strategy'.
2. Results section: ensure consistent use of 'Cas12a-hGEM' vs 'hGEM-Cas12a'.
3. Figure 3g: remove stray question mark after '0.6-fold?'.
4. Methods section: standardize units (µM vs uM) throughout.
5. Figure references: inconsistent capitalization of figure labels (Figure vs Fig.).

Significance

Overall, the manuscript represents an incremental, but meaningful advance rather than a conceptual reinvention of PCR tagging. The improvements described will be of clear practical value to users of the original method and to the broader genome editing community. Specific limitations of this study include the overreliance on a single cell line (HEK293T) as well as inherent methodological drawbacks, such as the toxicity associated with certain pathway modulation strategies and the likelihood of donor concatemerization. However, these limitations are clearly acknowledged by the authors and do not detract from the core contributions of the work.

I find this manuscript to be suitable for publication pending minor to moderate revisions. The technical advances are sound, the analysis is thorough, and the conclusions are well supported by the data. Addressing the major issues below, primarily related to scope, clarity, and contextualization, would further strengthen the manuscript and improve its accessibility.

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Referee #2

Evidence, reproducibility and clarity

Summary: In this study, Lou et al. provide an updated version of their previously established CRISPR/Cas12a-assisted PCR-tagging method for mammalian cells that supports large cassette integrations. They report improved fidelity and editing efficiency, introduce a strategy to quantify integration types using long read sequencing, and describe a one-step gene-tagging approach. The authors expand and improve upon their original protocol and analysis in the following ways: (1) The use of a tagging cassette that contains a tag-2A-marker fusion, allowing for easier selection of tagged clones. (2) The use of chemically modified PCR cassettes with phosphorothioate and biotin, which enhances the precision of integration. (3) Guiding cells towards HDR repair by chemical inhibition of NHEJ DNA-PKcs, particularly with inhibitor KU0060648. (4) Development of a Tn5-anchor-seq long-read sequencing platform coupled with a computational pipeline to classify knock-in outcomes such as HDR, NHEJ-mediated indels, concatemers, and off-target integrations. (5) A method for seamless tagging that better preserves endogenous 3'-UTRs by integrating tags immediately upstream of the target stop codon and removing the U6 cassette from the tag. Together, these developments have resulted in a method to incorporate large tags at endogenous loci with high efficiency. We expect this method will be a useful tool for researchers across many fields.

Major Comments:

• The most important shortcoming of this manuscript is the lack of biological replicates. The authors present results from multiple loci, but in this setting, variation will be largely driven by locus-specific effects rather than true experimental variation, which can be substantial for these types of methods. This makes it difficult for readers and potential users to evaluate how robust this pipeline is. The authors must present biological replicates for at least a small number of loci with their finalized method.
  • Significant time and resources will be needed.
• The authors characterize clonal genotypes following seamless PCR tagging at two loci. They report that 2/6 isolated clones of HNRNPA1 have the desired edit, while 3/3 clones of SLC16A1 have the desired edit. However, the small number of amplicons sequenced makes it difficult to draw robust conclusions about clonal genotype outcomes. The authors should perform additional amplicon sequencing to strengthen these conclusions. In addition, they should sequence the incorrectly sized products generated from the junction-PCRs to gain further insight into alternative genotypes and incorrect editing that may occur with this method.
  • Additional sequencing can easily be accomplished if cell lines are still available. An estimated time of about 1-2 weeks for cell thawing, genome extraction, amplicon sequencing, PCR purification, and outsourced Plasmidsaurus sequencing.
• It is not obvious how the authors were able to accurately quantify concatemeric integrations with the Tn5-anchor-seq tool. Specifically, the schematic in Figure 4a makes it clear that concatemers have multiple binding sites for the PCR primer. If these binding sites give rise to multiple products from the same template, the quantification would be inaccurate. The authors should comment on their considerations in suppressing the formation of secondary products, such as the use of a non-strand displacing polymerase, and in ensuring that their quantifications are accurate, including any bioinformatic steps.
  • The authors can clarify methods and adjust results presented in the manuscript in 1-2 days.
• The authors begin the results section by describing how their revised tagging cassette improves selection efficiency. It appears that they compare their newer data to data that were obtained for their previous manuscript, which were likely obtained at different points in time, potentially by different individuals, and using different sets of reagents, which makes direct comparisons impossible. It would be beneficial to include at least one side-by-side experiment to compare the authors' new method with their previous method to conclusively demonstrate any improvement.
• Data and method presentation: For much of the manuscript, the authors provide detailed descriptions of experiments and methods. However, the authors should provide more detail in the figure legends about the concentration and timing of chemical inhibitor treatments.
• Replication and statistical analysis: While the authors mention their lack of replicates, and reason multiple loci and conditions compensate for this, replication of their experiments is still necessary in order to draw meaningful conclusions of their data (see comment above).

Minor Comments:

References:

• The authors clearly identify and cite the prior work on which their method is based.
• The discussion focuses on the findings and limitations of the authors' specific study. Readers would benefit from a conceptual comparison of the strengths and weakness of their method to other similar technologies in this space, which would allow researchers to make better informed decisions about the appropriateness of this method for their own work.

Text and figures:

• The text and line of the scale bar on all microscopy images needs to be enlarged for readability.
• Text has inappropriate question mark in "... off-target frequency for TOMM20 by 0.6 fold?".
• Fold-changes in the text are described in an unconventional and unintuitive manner. For example, the authors note a 0.2 to 0.7-fold reduction in off target frequency contrasted against a 0.6-fold increase, which appear to be two different interpretations of fold-changes. These results might be more accurately represented by using a 1-fold change as the baseline for no change. Alternatively, the authors could provide this measure as percent decreases and increases.
• The authors mention that an example of diffuse cytoplasmic fluorescent signals observed in microscopy can be seen in Figure 2c, which are labeled with white arrows. A figure with this description is not present in the manuscript.
• Schematics in Figure 5 show mNeonGreen, however, Figure 5c is labeled as mScarlet. The authors should clarify which fluorescence tag was used.
• Figure S3e is mentioned in the text, however, the supplemental material does not contain a 3e.

Suggestions to improve presentation of data and conclusions:

• The authors have developed a fantastic pipeline to quantify tagging outcomes holistically, and in Figure 1f they visualize the results of the pipeline in an intuitive and informative manner. However, for the remainder of the manuscript, they proceed not to use this visualization and instead focus on the effects of their experimental parameters on individual outcomes or a ratio of two outcomes ("HDR purity"). This choice makes it difficult to clearly evaluate the effects of the parameters on all outcomes, and the "HDR purity" metric, although useful to compare against the gel-based readout, is difficult to interpret. We would recommend including the holistic visualization from Figure 1f for all experiments, in the supplement if necessary, for example as a large grid that depicts the frequency of all outcomes for each experimental condition by locus. This will make it much easier for readers to evaluate how a given experimental parameter affects the frequency of different editing outcomes.
• While the authors detail their rationale in selecting loci to tag in a previous publication, it is important to include the information in the current manuscript as well.
• The authors present many figures that look similar but display different quantities. To improve readability, they should use more descriptive figure titles and axis labels so that the main findings and quantities are immediately clear. This is particularly needed in Fig.2d, where the variable plotted is not clear.
• Two aspects of the final method may (or may not) represent limitations that would be helpful for the authors to discuss. First, the authors use a Cas12a with expanded PAM range, which has the advantage of increasing the number of available cut sites but also has the potential to lead to more off-target cutting, which can cause secondary mutations in the selected cell populations. Second, tagging the 3′ UTR may lead to differences in transcript and protein stability (especially due to the remainder of the 2A site). It would be helpful for the authors to include their perspectives on these issues and if they took efforts to address them.

Significance

Strengths/advance:

• This manuscript combines several established techniques into a single resource describing an updated PCR-tagging method for mammalian cells. In particular, the use of phosphorothioate and biotin-modified PCR cassettes together with the NHEJ inhibitor KU0060648 results in a pronounced increase in HDR while reducing indels, concatemers, and off-target integrations. This work takes CRISPR-assisted PCR-tagging from modest to strong editing outcomes and provides guidance and tools for others using this approach.
• Compared to other tagging methods in the field, this method allows for efficient integration of larger tags, such as an entire fluorescent protein, which is difficult to achieve using other methods.
• This study provides a useful method for cataloging and classifying integration outcomes with Tn5-Anchor-Seq plus a data processing pipeline.
• This study provides an updated online resource for designing primers for seamless PCR tagging.

Improvements:

• As the authors note in the discussion, demonstrating that this method is effective across cell models, especially physiologically relevant and/or therapeutically relevant models, would strengthen the impact of this work.

Audience:

• This study will be of interest to a broad audience, as improvements in mammalian CRISPR-mediated genome knock-ins are relevant to many fields.

Reviewer expertise:

• The PI and trainee together have expertise in genome engineering, synthetic biology, CRISPR technology development, and chemical screening.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The manuscript by Lou et al. present a technological advancement of a previous technique developed by members of the same laboratory (Fueller et al., 2020) that allows gene tagging of mammalian cells (HEK293-T cells) with PCR-generated DNA fragments containing the tag of interest, a selection marker, and a functional crRNA gene driving the integration of the whole cassette at the target locus. Their work focuses on the optimization of the original PCR-tagging protocol, which displayed sub-optimal efficiency in correct gene tagging, by introducing (i) additional design and experimental modifications (such as 2A-linked selection marker, chemical modifications of the PCR cassette, and pharmacological treatment of cells with ATR, NHEJ, or MMEJ inhibitors) and (ii) a further development of the Tn5-Anchor-Seq sequencing protocol optimized for long-read sequencing. As a result, they observed an overall improvement of the gene tagging efficiency by using chemically modified cassettes and NHEJ inhibitors, particularly regarding HDR (which is the desired repair mechanism) efficiency, and a reduction in the frequency of indel generation/concatemers/off-target integration. In addition, they show how their optimized PCR-tagging method can be used to obtain seamless integration, thus preserving the endogenous 3'-UTR of the tagged gene.

Major comments:

Although most results support the authors' conclusions, additional analyses are required for some instances that do not seem fully convincing, particularly related to unclear microscopy image analysis and related supporting data that are underrepresented in the manuscript, yet often referred in the text. More clarity about reproducibility should be also implemented. Specific comments: EVIDENCE:

1. In the first section of results, a self-cleaving 2A peptide is inserted upstream of the selection marker to improve selection of correctly tagged genes, as reported in the cited literature. To demonstate this in their PCR-tagging method, the authors tag the HNRNPA1 gene with the mNG-2A-NeoR strategy and find a tagging efficiency of 70% and more, in contrast with the 32% efficiency found with the original method developed by Fueller et al. (2020) (page 5). In support of this conclusion, only a representative image of cells showing mNG and HOECHST signal is presented, with no quantification (Fig1b). As this is a central result of the manuscript that allowed the authors to maintain the 2A-NeoR selection strategy for further investigation of sequence-level data, it is advisable to perform a more thorough quantification of the tagging efficiency by counting (with the help of image analyis software such as Fiji) the mNG+ cells and the total number of cells (HOECHST+), and calculate the % of mNG+ cells across different biological replicates (ideally, cells transfected in different days). A direct comparison (statistical analysis) with the % of mNG+ cells obtained by the original methodology (Fueller et al) would be desirable. In this way, the robustness of the improved tagging efficiency with the optimized method could be also evaluated within one single gene.
2. In the following line (page 5), authors talk about "correctly localized" signals, although no clear criterium of "correct" signal localization is explained, nor it is indicated anywhere in the example image. While in the following section(s) it is mentioned that diffuse signal may come from unspecific mNG expression of concatenated PCR cassettes, it would be still important to quantify the "correct" vs "uncorrect" mNG signal (i.e: nuclear vs cytoplasmatic) via image analysis.
3. Of particular significance would be to clarify whether there is a relationship between the % of concatemer product and the presence of diffuse cytoplasmic signal. In several instances, the authors mention their previous finding that diffuse cytoplasmic signal usually results from unspecific expression of the fluorescent reporter within a concatenated PCR cassette. However, from Fig1b there is no evident correlation between % of concatemer reads and % of diffuse cytoplasmic signal. Moreover, Fig2c is cited but no arrows are indicated in the figure in contrast with what mentioned in the text (page 10). Moreover, it is unclear what the authors would like to highlight in the following paragraph when talking about the percentage of diffuse signal (the discrepancy of the concatemers vs diffuse signal? Please make the paragraph more clear). Overall, I think that showing actual microscopy data and image quantification of diffuse vs localized signal would be ideal instead of only mentioning the result in the text. Moreover, a clearer and more detailed discussion about the authors' interpretation of the discrepancy observed between the % of concatemers and % of diffuse cytoplasmic signal is needed, and how it relates to previous findings by Fueller et al. (2020).

REPRODUCIBILITY:

It is clearly stated in the discussion that the study "[...] prioritized breadth (multiple loci and conditions) over depth (replicates) [...]", convincingly demonstrating the robustness of the technique to generally improve the tagging efficiency across genes tagged. However, I think that estimating the reproducibility of the improved methodology in tagging genes is important, as the authors are presenting a method that should allow researcher to easily tag genes in mammalian cells. While this does not necessarily mean that the authors should include biological or technical replicates to estimate the robustness of tagging a single gene in this improved method through sequencing, some actions are required to give indications to the reader about expected outcomes and to improve reproducibility by adding the following information: 1. It is expectable that the efficiency of PCR-tagging might vary if different preparations of the PCR products are used. The authors should add technical details in the method section that are currently missing regarding kits and protocols used for PCR, PCR purification/extraction, verification of PCR by sequencing (within the "Generation of plasmids and PCR cassettes"paragraph). This includes kits, polymerase type (Q5? Phusion?), extraction protocols (was the PCR gel-extracted?), sequence verification (was the extracted band sent for sequencing prior to perform PCR-tagging?) and so on. In particular, sequence verification by Sanger sequencing would help understand whether some small nucleotide changes were generated by PCR prior to cell transfection and should be added to the discussion part as source of possible mutations. 2. [OPTIONAL] A significant improvement of the study in terms of reproducibility would be to analyze, via microscopy image analysis (NOT by sequencing), the % of tagging efficiency as suggested above (see point 1 under the microscopy section) for the same gene (i.e. HNRNPA1 or other) with biological replicates consisting of cells transfected with PCR reactions prepared in different days, or [OPTIONAL] even analyzing how the tagging efficiency varies with PCR reactions purified with different kits or protocols (example: gel extraction vs PCR elution). Image analysis should include signal quantification with estimation of the % of tagging efficiency and statistical analysis. These experiment, not particularly demanding in terms of reagents and time (~3 weeks in total due to puromycin selection according to the workflow in Fig2b) would greatly help the reader understanding the robustness of tagging efficiency within single genes. 3. The concentrations of pharmacological inhibitors used (stated in the result section at page 8) should be included in the method section (in the "Transfection" section) and in FigS2, along with details of their use in cell culture. "If NHEJ inhibitors were applied, cell culture media containing small molecules (KU0060648, NU7441, VE-822, M3814) were added after 8 hours of transfection and cells were incubated for 16 hours. Two days after transfection, cells were selected with 500 μg/ml neomycin (Sigma-Aldrich)". Was the drug kept in the medium during neomycin selection? 4. While the authors mention the absence of technical/biological replication within single genes, some graphs are presented with error bars (Fig1F, Fig2d, FigS2b) although no details are given in the text. What replication has been done? What do the error bars represent? What is the sample size? 5. (pages 9-10, Result section): "We quantified the ratio of the HDR band intensity to the total intensity of both bands, defining this as "HDR purity". At the HNRNPA1 locus, we observed that both inhibition of c-NHEJ and synchronization of Cas12a increased HDR purity (Figure 3a)." How were the bands quantified to obtain the reported HDR purity %? Which software was used for band intensity quantification? Similarly, the information about how HDR purity was calculated with the anchor-seq data is lacking. These pieces of information are not present in the Method section and should be added.

Minor comments:

1. A few typos are present throughout the text. Examples: "because the probability of perfect insertion at both ends of the reporter are very low" (page 2); "With this 'mammalian PCR tagging' method constitutes a" (page 3); "In this work we address these gaps and employ selection markers fused in-frame to the tag of choice (e.g. mNeonGreen) using a 2A peptide spacing the two protein domains ensures that" (page 4); "but increased the off-target frequency for TOMM20 by 0.6-fold? (page 9); "from the wide-type and tagged cells" (page 17); "Together, our improvements outline an robust strategy" (abstract). Some other might be present, please check carefully.
2. The Acknowledgement part seems incomplete, and the author contributions are not listed.
3. Can the authors explain the concept/meaning of "index hopping" in the text (page 6)?
4. The FigS2g does not exist (cited at page 8). Did the authors mean f?
5. There is a discrepancy between Figures and labels regarding FigS3 at page 10: "The PS&Btn modifications on PCR cassettes reduced the frequency of concatemeric reads by 0.5- to 1.7-fold for most genes, while no effect was seen for CANX and SLC16A1 (Figure 4c and Figure S3d). As expected, inhibition of NHEJ with KU, NU, or M3814 decreased the concatemeric integration, while treatment with VE or the use of Cas12a-hGEM increased concatemer frequency at specific loci, such as CANX, DDX21, and SLC16A1 (Figure 4d, 4e, and Figure S3e). Inhibition of MMEJ by knocking down Polθ reduced concatemeric integration at the EMD and SLC16A1 loci but not at the LMNA locus (Figure S3e)." FigS3e does not exist, instead S3d seems to contain what S3e was supposed to show.
6. Figure 5b is never cited in the main text, nor elsewhere in the manuscript.
7. In the FigS2c caption, remove the sentence "All PCR cassettes used in this experiment were modified with PS and Btn." since both modified and unmodified PCR cassettes are shown in the panel.
8. The scale bar in the microscopy images (Fig1b, Fig5c) is not clearly visible, please fix the thickness.
9. In Fig5d, some lanes are highlighted in red although no explanation is given in the figure caption or in the text of what red color lanes represent.
10. In FigS1, different graphs of different panels have the same legend, that is placed only within panel f. The same applies for FigS3. It would help readability to state where to find the legend in the figure caption or to rearrange the panels so that the legend appears next to each graph.
11. Throughout the schematics used to exemplify the method workflow or cassette architecture there are several abbreviations that are not expanded in the relative figure caption. Examples: Figure 1a (HA, mNG, NeoR, U6, crRNA), Figure 1b (ONT, BC, Tn5) [...]. While some of them can be evinced in the main text, expanding each abbreviation in the respective figure caption can improve the figure readability.
12. In Fig1e, several symbols are present in the IGV snapshots whose meaning can only be guessed (what are purple vertical lines? What do numbers and letters indicate and what do the color indicate?). This information should be added as legend in the panel or explained in the figure caption. Similarly, it is not clear what the IGV snapshot indicates, it would be useful to add vertical lines in the IGV snapshot indicating, for example, the 5'-HA/3'-HA portion or something else that could help the reader understand what portion of the cassette is indicated.
13. Please specify that FACS was used only for the seamless tagging experiment, as it's unclear in the text. Were sorted cells seeded as one cell per well, or were they collected in one single tube and then separated? Why is Fig5b showing the sorting of mNG fluorescent cells while the result and method only mScarlet-i is mentioned (possibly wrong figure)?
14. In FigS2, panels e-f show the same variable (fraction of HDR reads). For EMD locus, however, there is a possible discrepancy on the fraction shown in e (yellow bar, PS&Btn) and f (grey bar, no treatment, modified oligos). Can the authors explain this apparent discrepancy?
15. Looking at some figures, such as Fig2, it is sometimes hard to relate the results to a "bigger picture". For example, Fig2d depicts "% of HDR integrations", while Fig2f shows "Fraction of HDR reads (%)". Besides possible confusion regarding the name of the variable analyzed, the caption of Fig2f explains that the relative panel contains the data in Fig2d. As Fig2d contains % of HDR-mediated integration of unmodified cassettes ranging 60-85%, one would expect to find the same percentages in Fig2f, under unmodified, control cassette; however, on average the fraction of HDR reads in these samples is only 10-20%. How is this possible? Is Fig2f including unmapped reads or reads mapped to off-target loci?
16. The graph in Fig3d displays Log2FoldChange of indel frequencies. To have a better understanding of the data, it is advisable to show an additional graph with "raw" indel frequencies for each gene, as side-by-side bars (similar to the graph in Fig2c). The same applies for the fold change shown in Fig3g.
17. [SUGGESTION TO IMPROVE CLARITY] Relative to the comments above (15-16), it would be advisable to add one or more graphs that summarize the read composition, similar to what has been done for Fig1f, for all genes analyzed in the different conditions. For example, it would be advisable to add a supplementary figure with one graph per gene analyzed (8 graphs in total) in the form of the one displayed in Fig2f, with stacked bars instead of boxplots as shown in Fig1f. This will help the reader understanding better and compare side-by-side the effect of the treatments applied in read composition for each gene.

Significance

The study provides an advancement in terms of tagging efficiency of genes in mammalian cells with design and protocol optimization of the PCR-tagging technique developed previously by the same lab, which suffered of low tagging efficiency. The study appears to bring a degree of novelty to the community interested in endogenous gene tagging of mammalian genes towards the goal of gaining a high efficiency in mammalian gene tagging, although representing a protocol optimization of a previously developed methodology. In particular, novelty is mainly technical, represented by the use of chemically modified PCR cassette, the introduction of P2A-linked selection marker, and the use of pharmacological inhibitors to suppress undesired repair mechanism in conjunction with improved computational pipeline.

GENERAL ASSESSMENT: The strength of the study is the thorough characterization of PCR-tagging and the step-wise protocol optimization analyzed with great detail at the sequencing level, particularly regarding the classification of possible outcomes. Following identification of the tagging outcomes exemplified in Fig1e, the authors introduce step-wise protocol optimization and characterize, for each integration outcome, if the added variables provide an improvement to the integration quality. Another strength resides in the analysis of multiple gene loci, indicating that the optimized tagging method can be used broadly. The matters described above about microscopy image display and analysis need to be improved, as several conclusions are drawn from such experiments throughout the manuscript. Moreover, a detailed explanation of some procedures central to the manuscript must be implemented, in order to improve reproducibility of the technique described.

ADVANCE: The study represents a technical advace of the previously developed PCR-tagging method. I had no prior knowledge of the PCR-tagging method or other similar methods used to tag endogenous genes in mammalian cells, so my evaluation of the advancement in the field is very limited, as well as whether the cited literature is comprehensive. Nevertheless, I was surprised to see that the Discussion section does not contain any reference (besides Fueller et al. 2020 in the first line), particularly regarding how the optimized method compares with other similar studies (for example Philip et al., 2025 and others cited in the introduction). While the authors do a great job in mentioning the possible limitations of the study and how to further improve the technique, most of the discussion focuses on general aspects of their study, failing (in my opinion) to contextualize the findings in the literature. Here, are only some examples of sentenced that can be contextualized a bit more: - "Likewise, transient suppression of Polθ (MMEJ) produced modest or gene-contingent improvements in our context, in line with emerging evidence that the relative contributions of c-NHEJ, MMEJ, and HDR vary with sequence architecture, chromatin environment, and cell state." - "This resolves a known ambiguity of selection based readouts: a cell can be drug-resistant and fluorescent for reasons other than a perfect on-target HDR event." - "Finally, selection based on a 2A-linked marker improves the fraction of functional fusions but could, in principle, bias against edits that affect translation or protein stability"

AUDIENCE: The study does not investigate the tagging efficiency robustness of single genes. Although this is a clearly stated choice by the authors, it results in being a resource mainly for labs interested in high-throughput gene tagging where the number of genes tagged are maybe more important than focusing on robustly tagging a single gene (the "breadth over depth" matter).

To conclude, I mostly enjoyed reading the work by Lou et al. As an experienced molecular and cell biology researcher currently working in genetic engineering and synthetic biology, I focused my attention in suggesting how to strenghten microscopy analysis and image display to support the author claims and help the reader understanding what changes in signal localization mean, as well as PCR-related methodologies. I also have prior experience in the production and analysis of sequencing libraries (RNA-seq in particular) and could evaluate that the presented analyses are well done and adequately detailed. I don't have sufficient expertise in editing mammalian cells to evaluate possible pit-falls of using pharmacological suppression of repair mechanism.

it shows that mapping isn't just about logging locations on a grid (see next page)

mapping can be utilized as a political tool that turns scattered urban gardeners into an organized social movement actively claiming their "right to the city." as Paula Restrepo writes.

It’s interesting to see that gap in how urban farmers think about sustainability, while composting and waste management are easy for people to connect to bigger environmental issues, way fewer growers see saving seeds as a direct pushback against our current economic system

Protecting "seed freedom" is an underrated part of urban sustainability overall

cities love to put urban agriculture onto low-income neighborhoods and call it "social justice," when in reality they’re just shifting the burden of food security onto poor residents instead of funding real structural change.

some people view urban farming purely as a hobby or gardening technique, while others see food sovereignty and agriculture as inherently political

Mapping out organic waste management is a smart move because it shows urban farming isn't just about growing food but it’s also about closing the resource loop and turning city scraps into compost to build a more circular local food system

Looking at these farms together instead of as isolated plots really changes their impact especially when linked up as a community network, urban agriculture goes beyond just growing food and becomes a way to push back against profit-driven city development

RHM used basic, accessible supplies like paper and glue to build this map, which shows that participatory urban planning doesn't need expensive GIS tech to accurately track local food networks and community assets

since urban agriculture is largely driven by low-income communities, local governments often leverage it as a dual strategy to address food insecurity and contain unregulated urban sprawl

Restrepo argues that modern cities prioritize capitalist profits over local farming, and is eseentially driving urban growth and industrial agriculture to dangerous levels

Dante’s The Divine Comedy describes nine circles of Hell, with each circle representing a different sin and punishment. The circles include Limbo, lust, gluttony, greed, anger, heresy, violence, fraud, and treachery. Each level becomes more severe, with the worst punishments reserved for people who commit the greatest sins. The passage explains Dante’s view of morality and the consequences of people's actions, using humor and modern-day examples to make the descriptions easier to understand.

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Note here that the editors of the volume are an architecture and urban studies professor (Bohn) and an eco-focused artist (Tomkins). Both based in the UK. The idea of "making visible" is interesting as a way of thinking about urbanity--analyzing and revealing existing metastructures (rather than designing new structures) as an architecture-adjacent practice.

Aha! (see my comments above)

The new ability to visually process huge amounts of data quickly and with minimal expertise means that purpose (see above) and creativity in choosing what and how to map are more central than ever

So a stakeholder takes initiative, while a citizen can be a passive consumer of food?

How to link past with present and future, without falling into the trap of nostalgia?

Might need to qualify this by explicitly defining "access". Do prisoners in a detention facility, students in a public school, or residents at a nursing home not have "access to food" even if they are being fed institutionally?

Similar to using positionality to frame an argument in academic writing. Mapping is a language/form of literacy as well

Her Infinite Cities series is a great example of creative urban mapmaking

A professor of architecture practice i heard at a conference forbade her students to use the word "stakeholder"--not because it is not a useful term, but because to her it somehow elides accountability to the heterogeneity of individual humans and more-than-humans

Again perhaps transgressing the map/territory distinction

So far, since the global financial crisis in 2008, middle class families have been found to have become just as precarious as poor working families.The author hints at a possible mass uprising. A similar uprising is the 1381 Peasants' Revolt where over 10,000 peasants rallied against the regime, led by a 14-year old king, because of the heavy flat-rate taxes imposed on everyone above the age of 15, following the Black Death. While the revolt was eventually quashed after less than a month, there was "no further levying of the poll tax for more than 250 years."

In Robinson's TMftF, revolts, just like terrorism take on a new meaning. Perhaps they have become one and the same. Are the Children of Kali rebels or terrorists? Whichever the classification, the fear they instill largely contributes to the reduction of Carbon levels.

I find it reckless to dismiss the Chinese style of pursuing prosperity because of the socio-political rivalry. Clearly, China's deep implication in the global economy must have something to do with its characteristically Chinese brand of Socialism. Perhaps this group may learn from that, tailor an ideology for themselves just like the Chinese, and prosper.

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A future chapter of TMftF mentions John Keynes. The central banks are indeed central to all of this because of Keynesian economics. John Keynes' emphasized the importance of government's intervention in the economy to deal with unemployment and to prevent inflation. The central banks' tools of trade for this are monetary policy and fiscal policy which are arguably the building blocks of the economy as we know it. They are used by the central banks and the federal reserve to influence demand and thus maintain stability.

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I think If you are lets say doing a TikTok dance made by a different person you could put in the caption DC(dance credits) and then the name of the person who created it. But I think in social media now a days as long as you are not copying someones video for verbatim you should be ok and not have to give any credit or citation.

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This book is so bad I'm quitting it.

You just introduced non unit circles....? So evaluate each of the following expressions for a unit circle?

I am not a fan of your book. This is not well-organized.

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we like winning as people