CRISPR-LMNA HDR assayU2OS were seeded in 6-well plates at 200 000 cells per well. Knockdown of PALB2 was performed 6–8 h later with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1.5 × 106 cells were pelleted for each condition and resuspended in 100 μL complete nucleofector solution (SE Cell Line 4D-Nucleofector™ X Kit, Lonza) to which 1μg of pCR2.1-mRuby2LMNAdonor, 1 μg of pX330-LMNAgRNA2, 1 μg of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct, and 150 ρmol siRNA was added. Once transferred to a 100 ul Lonza certified cuvette, cells were transfected using the 4D-Nucleofector X-unit, program CM-104, resuspended in culture media and split into 2 60-mm dishes. One dish was harvested 24 h later for protein expression analysis as described above while cells from the other were trypsinised after 48 h for plating onto glass coverslips. Coverslips were fixed with 4% paraformaldehyde and cells analyzed for expression of mRuby2-LMNA (indicative of successful HR) by fluorescence microscopy (63×) a total of 72 h post-nucleofection. Data are represented as mean relative percentages ± SD of mRuby2-positive cells over the YFP-positive population from 3 independent experiments (total n >300 YFP-positive cells per condition).