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Genome editing of donor-derived aβ+ T cells
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Figure 5. Evaluation of endonuclease specificity. (A) Putative off-target (OT) sites in the human genome (hg38) determined by COSMID and sorted by predicted rel- evance in descending order. Mismatches to the target site are marked red. OT 1-37 have three relevant mismatches in the protospacer region without insertions/deòetopms and OT sites 38-40 have two relevant mismatches and a protospacer-adjacent motif (PAM) mismatch. The nucleotide furthest from the PAM was ignored for sorting due to mismatch tolerance at this location by Cas9. (B) Human T cells from six different donors were electroporated with the high-fidelity Cas9 protein complexed with the sgRNA targeting the TRAC locus (or mock electroporated to determine background). Next-generation sequencing was performed on all predicted OT sites. The dotted line depicts the sensitivity limit attributed to this method of 0.1%. sgRNA: single guide RNA, PAM: protospacer-adjacent motif; OT: off-target.
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