Chapter 4 —— 68 —— specificity eCas94NLS:GS1 or regular Cas9:GS1 nucleases. (B) Establishing AAV gene targeting. EGFP-sorted hMSCs genetically modified via the delivery of AAV-HRS1 together with high-specificity eCas94NLS:GS1 or conventional Cas9:GS1 nucleases, were subjected to junction PCR assays detecting HDR-derived telomeric and centromeric junctions (jT and jC, respectively) (n = 2 biological replicates). (C) Assessing AAV gene targeting specificity. EGFP-sorted hMSCs genetically modified through the transfer of AAV-HRS1 together with high-specificity eCas94NLS:GS1 or regular Cas9:GS1 nucleases, were subjected to junction PCR assays specific for HDR-independent insertions at the validated top-ranked gRNA GS1 off-target site at CPNE5. The regular Cas9:GS1 complexes were applied using two total doses of AdVs, i.e. 32 and 64 GC cell-1; the high-specificity eCas94NLS:GS1 complexes were applied at 20 GC cell-1. (D) Assessing eCas94NLS:GS1 nuclease-induced DNA cleaving specificity. hMSCs were transduced with AdVP.eC94NLSGS1 at the indicated MOI and DNA cleaving at the AAVS1 target site and at the highest and intermediate-ranked validated off-target sites in CPNE5 and BBOX1 loci, respectively, were quantified by next-generation deep sequencing at 3 days posttransduction (∼50000 paired-end reads per sample). Mismatches between the spacer of gRNA GS1 and off-target DNA sequences within CPNE5 and BBOX1 alleles are highlighted in red. Histograms corresponding to the types and distributions of insertions and deletions (indels) at AAVS1 established after NHEJ repair of DSBs induced by eC94NLS:GS1 complexes are also plotted. (E) Experimental setup for experiments in HeLa cells. HeLa cells transfected with plasmids encoding regular Cas9:GS1 or high-specificity eCas94NLS:GS1 nucleases or eCas94NLS:GS1 nickase, were transduced with donor AAVHRS1 at 1.0 TU cell-1. After sub-culturing and EGFP-directed cell sorting, stably transduced HeLa cell populations were subjected to off-target AAV donor insertion analysis by CPNE5-targeted PCR. (F) Off-target AAV donor DNA insertion analysis. Schematics of AAV donor-derived sequences inserted through non-homologous endjoining processes at the major off-target site of gRNA GS1 at CPNE5, are depicted (left panel). PCR analysis using a primer pair flanking the major off-target site of gRNA GS1 at CPNE5 (right panel). Open arrowhead mark CPNE5 sequences without donor insertions; EGFP served as an internal control template for availability and integrity of transgenic DNA (n = 2 biological replicates). gene expression due to their intrinsic promoter activity [66,67]. Notably, high-throughput deep sequencing established a direct correlation between the detection of donor DNA insertions at CPNE5 (Figure 7C) and DSB formation at this major gRNA GS1 off-target locus (Supplementary figure
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