Dual Barcode is Essential for the Specificity of DSB Repair Capture
We discovered that PCR-mediated template switching poses a significant challenge in ensemble tagged PCR, particularly in the template sequences with high similarities, which we successfully addressed by introducing a dual barcode. Template switching presents in repair sequencing (repair seq) and severely interfere the interpretation of the association between repair outcomes and the sgRNA in the vicinity. Among the 67 DNA damage response genes, we found that ERCC6L2 was crucial for preventing the DNA end resection. Overall design: K562-iCas9-DDR cell line was generated by transfection lentivirus bearing iCas9-blast (includes GFP) and select a K562-iCas9 cell line, followed by transfecting lentivirus bearing pMCB320-DDR bear dual barcodes (includes mCherry) and selected in RPMI-1640 with 5 µM puromycin plus 5 µM blasticidin for two weeks until mCherry-positive cells reached > 99%. To knockout the corresponding DDR genes, 2 µM Doxycycline was added and maintained for more than one week. The DSBs in cycling and G1/G0-arrrest K562-iCas9-DDR cells were generated by Cas12a plus DDR1-crRNA nucleoproteins (63 pmol Cas12a:crRNA per 2 million cells) targeting a region in the dual-barcode DDR cassette. After nucleofection, the K562-Bcl2 cells were maintained in RPMI-1640 plus 5 µM puromycin and 5 µM blasticidin for two days. The genomic DNA were extracted and subjected to standard library prepration with two rounds of PCR.