Figure 5.

Off-Target Site Analysis

(A) Potential off-target cleavage hits for the gRNA N1 were identified by ZiFiT Targeter 4.2 in the human genome. Mismatches (MMs) are underlined. Note that there is no off-target with 1 MM. 3 potential off-targets were found with 2 MMs in non-coding regions. In addition, 3 potential off-targets with 3 MMs and 2 potential off-targets with 4 MMs were identified in coding regions. The 8 off-target sequences were subjected to the Surveyor cleavage assay. (B) Genomic DNA from RDEB-Ks treated with IDLVs encoding for the LV-CRISPR-N1 (2 pg p24 per cell) was extracted, and regions corresponding to off-target sequences were amplified by PCR using specific primers (listed in Table S4). The Surveyor cleavage assay was performed at each potential off-target hit. No Surveyor activity indicative of cleavage at predicted off-target sites was detected. nd, non-detected.