Next-Generation Sequencing (NGS) and pooled screen analysis
Plasmid DNA was isolated using a Plasmid Maxiprep Kit (Biomed). Integrated guide RNA target pair sequences were PCR-amplified using Q5 polymerase (NEB) for frequency analysis. PCR products were purified using a QIAquick Purification Kit (QIAGEN). For each sample, 1 microgram of purified PCR amplicons were used as a library template. Sequencing libraries were obtained from the replicates using a NEBNext Ultra II RNA Kit (NEB)and purified with SPRI beads(Beckman). The sequencing libraries were prepared following the manufacturer’s protocol(Illumina).Library size and purity was verified by Agilent 2100(Agilent) before sequencing on a Nova seq (Illumina) using a Reagent Kit S2 (Novaseq) (2 × 150 bp).