Figure 2: A germline SNV creates a single high-efficiency off-target site. | Nature Communications

Figure 2: A germline SNV creates a single high-efficiency off-target site.

From: Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells

Figure 2

(a) A heterozygous-common SNV at the Chr5_OT site in the genome of PGP1 cells creates a genomic allele with only two mismatches to the TAZ-targeting gRNA (Chr5_OTvar). Chr5_OTvar was targeted in both TAZ-clones, as evidenced by the small-deletions detected by WGS, while the reference allele Chr5_OTref with three mismatches remained intact. (b) Deep sequencing of 31 (out of 32) potential off-target sites with at the most three mismatches revealed Chr5_OT as the only high-efficiency off-target site. Sequences of the predicted off-target sites and the indel frequencies for each site can be found in Supplementary Tables 1 and 2. (c) Deep sequencing revealed the allele-specific nature of Cas9. Cas9 demonstrated high-targeting efficiencies at the TAZ-target site and the Chr5_OTvar allele but minimal efficiency at Chr5_OTref as measured by the indel frequency. The indel frequency at Chr5_OTref was in the same range as indel frequencies at all other three-mismatch off-target sites (Supplementary Table 2). (d) Varying the gRNA sequence relative to the target-site sequence confirmed the high-mismatch sensitivity of Cas9. The pairwise targeting efficiency between each gRNA and each target site was measured by the indel frequencies detected by deep sequencing. Relative efficiencies were calculated by normalising the off-target indel frequencies to the on-target frequencies.

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