Deep sequencing data were analyzed using in-house Python scripts (Supplementary Code), which were modified from previously used code (22). Each guide RNA and target sequence pair were identified using the unique 15-nt barcode sequence located upstream of the target sequence. Insertions or deletions located around the expected cleavage site (i.e., the 8-nt region centered on the middle of the cleavage site) were considered to be Cas9-induced mutations. To exclude the background indel frequencies originating from array synthesis and PCR amplification procedures, we normalized the observed indel frequency by subtracting the background indel frequency determined in the absence of Cas9 delivery according to the functionIndel Frequency (%)=Indel read(Total read × background indel frequency)Total read(Total read × background indel frequency) × 100

To increase the accuracy of the analysis, deep sequencing data were filtered; target sequences with deep sequencing read counts below 200 and background indel frequencies above 8% were excluded as similarly performed previously (21).

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