title: Genome-wide CRISPR knockout library screening coupled with high-throughput sequencing to analyze DNA repair outcomes
design: Genome-wide CRISPR knockout library screening coupled with high-throughput sequencing to analyze DNA repair outcomes
library_construction_protocol: The human GeCKOv2 lentiviral library (GENEWIZ, Suzhou), containing 122,756 sgRNAs targeting 19,050 genes (6 sgRNAs per gene), 1,864 miRNAs (4 sgRNAs per miRNA), and 1,000 NonTarget controls, was transduced into 3 × 10⁸ HEK293T cells at an MOI of 0.3. After 24 hours, cells were selected with 1 μg/mL puromycin. After selection, surviving cells were expanded for 4 days, the resulting IPGRM cell pool was subjected to secondary editing. In brief, a total of 6.3×107 IPGRM library cells were seeded into three T175 flasks 24 hours before transfection and cultured to 70-80% confluence. Each flask was co-transfected with 7.5 μg pCMV-Cas9 and 15 μg pU6-sgRNA plasmids targeting either sg3 or sg81 Genomic DNA was extracted using MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa).  PCR amplification were performed in 20 μL reactions containing 40 μg genomic DNA, 0.4 μM of each primer, and Q5 Hot Start HiFi PCR Master Mix (NEB). Cycling conditions were: 98 °C for 2 min; 20 cycles of 98 °C for 10 s and 65 °C for 45 s; and a final 5 min extension at 65 °C. PCR products were purified using Cycle Pure Kit (Omega) and size-selected by Gel Extraction Kit (Omega).