DNA binding proteins recognize and bind with specific DNA sequences. They are useful not only in genomic editing but also in regulation. http://www.wiley.com/college/pratt/0471393878/student/structure/dna_binding_proteins/index.html

See the supplementary material for details about how the authors did this.

This is another area for potential future research

This is another area for potential future research.

Other genes derived from a common ancestor

This is a problem that will definitely need to be resolved before these technologies can be deployed in human beings

This is another area for possible future research.

This could be an area of future research

This is crucial to the bacteria or archaea distinguishing between self and not self.

These are the two existing commonly used genetic editing strategies.

These tables are available for future researchers who wish to use the method described here.

The authors ran a computer simulation to see how much of the human genome could possibly be targeted using this system.

The authors had previously inserted DNA into an artificially inserted target. Now they targeted a natural site.

Puromycin is an antibiotic that can be used to select cells which have developed resistance to it by killing off all the cells which have not. It is used here to purify a cell strain by eliminating all the cells which did not develop resistance. http://agscientific.com/blog/index.php/2012/10/10/puromycin-faqs/

Sanger sequencing is a method of reading DNA codes which relies on normal and modified deoxynucleosidetriphosphates (dNTPs). The modified dNTPs terminate the sequence prematurely, and the sequence is then assayed https://www.thermofisher.com/us/en/home/life-science/sequencing/sanger-sequencing/sanger_sequencing_method.html

This is a method for rapidly increasing the amount of a sample of DNA by artificially replicating the DNA using a polymerase.

A DNA sequence derived from self.

This article describes current state of the art in the use of transcription activator-like effectors (TALES) in genetic editing.

This article reports the use of a green fluorescent protein reporter system in a project to edit the gene related to sickle cell anemia.

This article focused on research involving transferring the Staphylococcus thermophilus CRISPR3 system to Escherichia coli.

This article zeroes in on the CRISPR system of Streptococcus thermophilus, the system which was chosen by the authors of the present article.

This article reports research on the mechanisms of the type II CRISPR system which paved the way for the research described in the current article.

This review focuses on the function of the CRISPR system http://www.micab.umn.edu/courses/8002/Terns.pdf

This review explains both the function of the CRISPR system and current genetic editing research in the area. http://dpb.carnegiescience.edu/sites/dpb.carnegiescience.edu/files/Bhaya_ARG.pdf

This review explains what is currently known about how the CRISPR system functions.

Editing using more than one targeting strategy.

The deletions were not always exactly on target, but they tended to center around the target thus providing evidence that the process itself targets specific DNA sequences.

That is, the AAVS1 locus in the PPP1R12C gene of chromosome 19 as mentioned above.

In non-homologous end joining splices in DNA are repaired by splicing rather than by referring to an intact complementary strand of DNA. This method of repair is less accurate than homologous recombination.

This is the integrated reporter mentioned earlier in the paragraph.

The authors checked the DNA sequence of daughter cells after they did the editing

Induced pluripotent stem cells come from adult cells which have been artificially changed to exhibit stem cell properties. These properties are immortality and the ability to develop into various different cell types. These cells are thus unspecialized. https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/generation-human-induced-pluripotent-stem-cells-fibroblasts.html

This cell line is derived from myelogenous leukemia cells and is considered an excellent target to test the function of natural killer cells. http://www.atcc.org/products/all/CCL-243.aspx

These cells are the human embryonic kidney cells mentioned above.

These are genes which are expressed in almost all the cells or an organism.

Chromosome 19 includes about 1500 genes and 59 million base pairs. For a brief summary of chromosome 19 see http://ghr.nlm.nih.gov/chromosome/19

This is a gene concerned with protein phosphatase regulation http://www.genecards.org/cgi-bin/carddisp.pl?gene=PPP1R12C

This is a site on human chromosome 19 which the adeno associated virus type 1 targets.

The researchers now turned to modifying a natural DNA sequence.

The integrated reporter was the green fluorescent DNA sequence which was integrated into the target DNA. The green fluorescent sequence could then be manipulated, and the results could be measured.

The authors demonstrated that the system can edit various different genes in the human genome.

http://www.genecards.org/cgi-bin/carddisp.pl?gene=DNMT3A

This supports the authors' interpretation of what is happening.

DNA strands have 2 ends, a 3' end and a 5' end. The 3' end is joined to the 5' end of the complementary strand.

By increasing the proportion of homologous recombination the researchers have better control over the process. Nonhomologous end joining is a less exact process and leads to more spontaneous errors.

This is an enzyme which nicks specific sites in the genome without damaging unintended sites.

This provides support for the authors' description of the mechanism for the system earlier described.

The results of the editing process appeared sooner in the case of the authors' engineered guide RNAs as compared with the earlier method using TALENs

In other words, 3-8% of the cells returned to being fluorescent.

Heterodimers are molecules composed of two different macromolecular chains.

The authors constructed two different guide RNAs

This is the 68-bp fragment mentioned toward the end of the last paragraph. When it is expressed, it makes the protein non-fluorescent.

A transcription activator-like effector nuclease is an artificial restriction enzyme (enzyme that cuts DNA at a specific spot). Talens are created by fusing DNA binders to DNA cleavers. They are modeled from proteins secreted by Xanthomonas bacteria. http://bfg.oxfordjournals.org/content/early/2014/06/06/bfgp.elu013.full

The authors generated a database of about 190,000 guide RNAs which can potentially target about 40.5% of the human genome. This will be a resource for future experiments.

Multiplex editing aims either to make several different changes at once or changes in several different locations of the genome at once.

The authors had to show that the results follow the addition of specific components to the system. By demonstrating this, they support their explanation for what is happening on the microbiological level.

This is a microscopic process which cannot be directly observed, so it is important to show that the process really does rely on CRISPR components.

Exons are parts of genes that code for proteins.

This method uses a laser to identify which cells are of interest and thus sort them out. http://www.bio.davidson.edu/courses/genomics/method/facs.html

Homologous recombination is a naturally occurring method of repairing breaks to DNA. This method relies on the unbroken strand of DNA to correct errors on the broken strand. https://www.qiagen.com/us/shop/genes-and-pathways/pathway-details/?pwid=143

By introducing the green fluorescent protein sequence and then disrupting it the authors were able to measure how well their genetic editing worked. They could measure this by determining how many of the daughter cells were fluorescent.

This is a site on human chromosome 19 which the adeno associated virus type 1 targets.

Since DNA consists of a double strand, the bases form into pairs such that cytosine always pairs with guanine and adenine always pairs with thymine. A 68 bp sequence would contain 68 of these pairs.

This means that the green fluorescent protein is integrated into the host's DNA

A stable cell line is one in which there is little variation from one cell to another. Another desirable characteristic of stable cell lines is that most of the cells are not actively dividing unless they are stimulated to do so by the researcher. https://www.mirusbio.com/tech-resources/tips/generate-stable-cell-lines

These cells were developed from the kidney of a human embryo. They are easy to grow, and it is easy to introduce foreign nucleic acids (RNA and DNA fragments) into them. http://www.atcc.org/products/all/CRL-3216.aspx#documentation

Green fluorescent protein is naturally produced by the jellyfish species Aequorea victoria. It can be used to tell whether or not a gene has been expressed . (Expression means that the characteristic of the gene appears in the phenotype of the organism). https://www.jic.ac.uk/microscopy/more/T5_9.htm

The system can target sequences between one guanine (G) followed by 20 nucleotides (pairs of amino acides) and ending with two guanines in a row (GG).

The U6 gene is commonly found throughout the human genome. It is also a well studied gene

The PAM is a short DNA sequence close to the targeted DNA sequence of the invading organism. The PAM is crucial for the system to recognize whether or not the DNA sequence is self or not self. If the PAM is missing the system will not be activated

An enzyme important in the production of short RNA

In the natural CRISPR system guide RNAs take fragments of the viral or plasmid DNA and guide them to specific locations in the CRISPR locus. The idea here is that these artificial guide RNAs can similarly guide a short strand of nucleic acids into a specific place in the target genome.

The authors combined two proteins which are needed for correct functioning of the CRISPR type II system.

Genes code for proteins, but characteristics of the cells that the genes are in may either help or hinder the genes from actually producing the desired protein.

For example, there are differences between insect protein and the protein of mammals. Cells can be engineered to produce the desired type of protein. For more about expression systems, see this link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848218/

Nuclear localization signals are amino acid sequences that mark a protein to be transported to a specific place in the nucleus.

CRISPR systems provide bacteria and archaea with acquired immunity and may also play a role in regulation of DNA expression. For a review of CRISPRs, see this link:https://dpb.carnegiescience.edu/sites/dpb.carnegiescience.edu/files/Bhaya_ARG.pdf

The Cas9 protein is the hallmark protein of type II CRISPR systems

A codon is a sequence of DNA "letters" that corresponds to a specific amino acid. By using a sequence of codons, specific proteins can be constructed.

The bacterial type II CRISPR system is the best known system and also the least complex system.

Genome engineering is targeted change in the DNA sequence.

In contrast to DNA, which is a double strand of linked amino acids, RNA is a single strand. The four "letters" in the DNA alphabet are adenine, thymine, cytosine, and guanine. RNA is made up of adenine, thymine, and cytosine, but in place of guanine it has uracil.

Breaking

Eukaryotic cells have a membrane around their nucleus, in contrast to prokaryotic cells such as bacteria or archaea.

The cited article describes an experiment which demonstrated DNA cleavage by Cas9, principal protein in type II CRISPR systems. Cas9 protein was purified from a preparation of Streptococcus pyogenes.

The researchers found that addition of crRNA alone was not able to guide cleavage of target plasmid DNA, but with the addition of tracrRNA the cleavage occurred.

By comparing these results with similar results using short double sequence DNA, the authors demonstrated that the functions of tracrRNA are triggering the enzyme RNase III and activating cleavage of target DNA by means of cas9 under the guidance of crRNA.

The authors fused the two essential elements: crRNA and tracrRNA to provide greater convenience for targeted genetic editing.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science (Washington D C), 337(6096), 816-821.

Deoxyribonucleic acid (DNA) is a double stranded helix (coiled in a spiral) of proteins which form a sort of "alphabet" to encode genetic information. The "letters" of this alphabet consist of adenine, thymine, cytosine, and guanine.

Adenine always pairs with thymine, and cytosine always pairs with guanine, thus, when the two strands are divided the complete DNA molecule can be exactly reproduced. http://ghr.nlm.nih.gov/handbook/basics/dna

Plasmids are circular double stranded DNA fragments which can insert themselves in a cell and thenceforth be reproduced in cell divisions even while staying apart from the cell's own DNA. Plasmids can spread resistance to antibiotics from one strain of bacteria to another. They are also important for genetic engineering because they can be used for targeted DNA changes. http://www.nature.com/scitable/definition/plasmid-plasmids-28

Viruses are submicroscopic parasites with a simple structure. Their main parts are a strand of nucleic acid (DNA or RNA) and an surrounding protein shell called a capsid. Some viruses also have an outer viral envelope surrounding their capsid. Viruses are not capable of reproducing on their own, but rather must invade a cell and commandeer that cell's resources to reproduce themselves http://www.ncbi.nlm.nih.gov/books/NBK21523/

CRISPR associated proteins are proteins which act in concert with the rest of the CRISPR system to mediate its activities. These proteins are grouped in families. The types of proteins present characterize the distinction between type I, type II, and type III CRISPR systems http://www.biologydirect.com/content/6/1/38

CRISPR RNAs are short strands of RNA transcribed from the CRISPR loci which play an important role in targeting foreign DNA https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

Induced pluripotent stem cells are cells derived from adult tissue which have been artificially made to act like stem cells. In other words, they have been forced to be capable expressing the full range of their gene capacities instead of specializing as adult cells normally do http://stemcells.nih.gov/info/basics/pages/basics10.aspx

293T cells were originally derived from the kidney tissue of a human embryo. They have been modified to contain the SV40 T antigen, thus giving this cell line advantages for work with retroviruses. http://www.atcc.org/products/all/CRL-3216.aspx#characteristics

SV40 T antigen is associated with Simian Virus 40, a tumor producing virus which is in the Polyomavridae family http://www.nature.com/onc/journal/v24/n52/full/1209046a.html

293T cells were originally derived from the kidney tissue of a human embryo. They have been modified to contain the SV40 T antigen, thus giving this cell line advantages for work with retroviruses. http://www.atcc.org/products/all/CRL-3216.aspx#characteristics SV40 T antigen is associated with Simian Virus 40, a tumor producing virus which is in the Polyomavridae family http://www.nature.com/onc/journal/v24/n52/full/1209046a.html

The AAVS1 locus is a target on human chromosome 19 that is frequently used for testing purposes. The reason for this is that there have been no known adverse effects from genetic manipulation in this area http://www.genecopoeia.com/product/aavs1-safe-harbor/

Guide RNA is a genetically engineered fusion of crRNA and tracrRNA. It targets the RNA sequence of interest and enables it to bind with the Cas9 nuclease https://www.addgene.org/CRISPR/guide/

There are three types of CRISPR systems: Type I, II, and III. Type II is the simplest type and is characterized by an operon (functional DNA unit) of only four genes: cas9, cas1, cas2, and either cas4 or csn2. https://dpb.carnegiescience.edu/sites/dpb.carnegiescience.edu/files/Bhaya_ARG.pdf

The scientists who first discovered these sequences had no understanding of their possible function. Later it was observed that some of the spacers correspond to the DNA of invading viruses.

This led to the discovery that CRISPRs play a protective role defending bacteria and archaea from other invasive organisms.

There is speculation that this immune function is only one of the functions of CRISPRs. They may be involved in other functions such as regulating the genetic expression of the barcterial or archaeal organism's own DNA. https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/

Clustered regularly interspaced short palindromic repeats (CRISPRs) are strands of DNA with repeating sequences (repeats) interspersed with other sequences which don't repeat (spacers).

CRISPRs do not act in isolation. They are part of a complete system which includes the CRISPR array, associated genes, and an upstream leader sequence. The CRISPR array is the strand of DNA with repeating and unique segments interspersed.

Many, but not all, of the repeating segments are palindromes (sequences that read the same from right to left as the do from left to right). Many of the spacers have DNA which matches that of invading viruses (phages) or plasmids. CRISPR associated genes often code for proteins which can cleave or bind DNA or RNA sequences. The leader sequence of the system occurs before the first repeat and is important for transcription as well as for obtaining new spacers. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497052/

Adaptive immunity is immunity that one gets in response to challenges from the environment. This is in contrast to innate immunity, which is the immunity one is born with.

Adaptive immunity is not something exclusive to higher animals. Bacteria and archaea also have immune responses to foreign organisms which threaten them (mainly viruses and plasmids) http://www.ncbi.nlm.nih.gov/pubmed/23495939

Like bacteria, archaea are also prokaryotes. Archaea have similar appearance and behavior to bacteria but differ greatly from bacteria in their genetic structure. Many archaea live in extreme environments such as extremely hot or cold areas or areas with high salt concentrations http://www.microbeworld.org/types-of-microbes/archaea/42-what-is-a-microbe-sp-828/types-of-microbes/149-archaea

Bacteria are prokaryotes. This means that they don't have a nucleus to contain their DNA. In contrast, eukaryotes have a nucleus which contains their DNA. http://www.microbeworld.org/types-of-microbes/bacteria

Fully defined systems are standardized. Individual variations are minimized, thus enabling to system to yield predictable results.

In vitro means "in glass". This is used to refer to an experiment conducted in laboratory containers rather than in a natural environment

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What are the differences between bacteria and archea? Link Text