Targeted Deep Sequencing of Genomic Edits

Eight thousand sorted cells were harvested for genomic DNA extraction by addition of 20 μl of lysis buffer (Vazyme) following the manufacturer’s manual. For TIDER test, the genomic region in the vicinity of Cas nuclease target site was amplified by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) using nested PCR. Purified PCR products were Sanger sequenced and analyzed as previously described^{44 (link)}. For deep sequencing analysis, the targeted genomic region was amplified by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) using nested PCR, primers with barcode were used. PCR products were purified by Gel extraction kit (Vazyme) and sequenced on an Illumina HiSeq X System (150-bp paired-end reads). Forward reads were aligned to the reference sequences using BWA (v0.7.17-r1188) with parameter of “bwa mem -A2 -O3 -E1”. At each target, editing was calculated as the percentage of total reads containing desired edits without indels within a 10-bp window of the cut site. The target site informations are provided in Supplementary Table 2.