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We next assessed how reflective the HDR editing
We next assessed how reflective the HDR editing rates calculated via deep-sequencing were of actual editing rates. Hence, the CTS protocol was repeated for all seven variants independently, and approximately 250 clonal populations were then picked and analyzed via Sanger sequencing. We successfully isolated cell lines for all seven mutations and observed a concordance between the quantities of HDR editing events determined via deep-sequencing and direct sequencing (Figures 3 and S3; Tables S2 and S3). We note that pooled deep-sequencing measures allelic representation at a specific time point and does not reflect zygosity or account for differing cellular growth rates from which the pool was derived. Extent of heterozygosity seemed to correlate, at least for the CRYAB mutations, with increasing distance from the CRISPR/Cas9 cut site (i.e., increased observation of heterozygous knockin clones with CRYAB:c.325G>C), but not in the case of BAG3:c.1430G>A, where we failed to isolate any mutant homozygous lines even though the targeted nucleotide was immediately adjacent to the CRISPR/Cas9 cut site. We speculate, as others have, that the nature and extent of zygosity is dictated by the distance between the CRISPR/Cas9 cut site and the targeted nucleotide as well as locus-dependent factors, such as buy Cyclopamine organization (Paquet et al., 2016; Ward, 2015; Yang et al., 2013).
We analyzed the zygosity and specificity of pac cassette knockin at the AAVS1 locus by Southern blotting and an integration-specific PCR assay (Figures S4A and S4B). We found that 50% of the clones (24/48) were correctly targeted without additional random integration. Of these, 21/24 were heterozygous and 3/24 were homozygous (Figure S4C). We observed concordance between results from Southern blotting and the PCR-based assay. With the PCR-based assay, representative cell lines harbored the pac cassette at AAVS1 (Figure 3) and none displayed evidence of additional aspecific integration events (data not shown). In addition, all lines analyzed exhibited the typical pluripotent cell morphology and karyotypic stability as well as expression of pluripotency markers (Figure 3), and maintained a capacity to form high-representation cardiomyocyte cultures (data not shown).
Despite improvements in sgRNA design (Doench et al., 2016), we nevertheless analyzed the top potential off-target sites (Experimental Procedures) in multiple mutant cell lines via Sanger sequencing and detected no signs of aspecific cleavage (data not shown). Furthermore, deep-sequencing of potential off-target sites (Experimental Procedures) revealed that CTS did not enrich for aberrant cutting compared with unselected cells (data not shown). The TALENs targeting AAVS1 have been previously demonstrated to have minimal off-target cleavage (Hockemeyer et al., 2011). Collectively, these data suggest negligible reagent promiscuity and that while CTS enriches for cells bearing HDR-edited alleles, it does not enrich for off-target mutations. We note that GUIDE-seq (Tsai et al., 2015) or similar would be required for whole-genome examination of off-target cutting.
CTS is an inexpensive, rapid, straightforward, and readily scalable method, conceptually analogous to other marker-assisted enrichment strategies (Arribere et al., 2014), which increases the likelihood of isolating cells bearing knockin alleles by providing a non-integrating reporter of the HDR pathway activity akin to that devised by Flemr and Buhler (2015). While the precise cellular mechanisms of ssODN-mediated double-strand break repair remain to be elucidated, the observed enrichment by CTS via canonical homologous recombination of the double-stranded pac cassette donor into AAVS1 suggests a transient state of HDR permissiveness and a potential overlap in these repair mechanisms in iPSCs. We encountered significant variability in inter-locus targetability and suspect that local sequence composition and chromatin organization likely influence repair preference. Given the multifactorial nature of complex diseases and especially the role of modifier loci, we envisage CTS being of potential utility for simultaneous recapitulation of multiple candidate variants. Indeed, using CTS, we concurrently delivered editing reagents designed to incorporate passenger mutations at two different loci and isolated multiple clones bearing both edits (Figure 4). It will be interesting to determine whether application of CTS in conjunction with polycistronic sgRNA delivery systems (Cong et al., 2013) will permit highly parallelized HDR-based genome editing.