Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…24,[33][34][35][36] Previous studies on a limited number of human CRISPR/Cas9-edited PSC clones reported a low incidence of offtarget mutagenesis as detected by whole-genome sequencing, but they concluded that these changes were most likely not related to CRISPR/Cas9 editing, due to low sequence similarity between the off-target indels and the intended target sites. 37,38 However, a recent study using deep sequencing to detect off-target editing of human AAVS1-T2 gRNA, the counterpart of our rhesus AAVS1 gRNA, found a significant number of indels at several endogenous loci in 293T cells and one site in a targeted human iPSC line. 39 In the current study, we identified potential off-target sites in the rhesus genome using the same algorithm, 24 and indeed we found indels at one or two sites in half the tested clones.…”

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

“…The sgRNA/Cas9 complex recognizes target sites based on the rule of Waston-Crick base pairing, conferring overall high specificity of the CRISPR/Cas9 system Smith et al, 2014;Veres et al, 2014). However, CRISPR/Cas9 was found to tolerate MMs between its sgRNA and target sites (Duan et al, 2014;Kuscu et al, 2014;Lin et al, 2014b).…”

“…Among the total 45 top-ranked off-target sites (15 for each of the three most potent sgRNAs) tested, we observed no significant OTEs, confirming the specificity of the CRISPR/Cas9 system. However, due to the limited sites we analysed and the restricted sensitivity of the T7EI cleavage assay, lower incidents of OTEs for these sgRNAs at a genome-wide scale remain to be determined by more powerful approaches, such as ultradeep next-generation sequencing of the whole genome Smith et al, 2014;Veres et al, 2014). Studies aimed to develop modified CRISPR/Cas9 systems are also promising to further improve the on-target fidelity of CRISPR/Cas9 (Guilinger et al, 2014;Qi et al, 2013;Ran et al, 2013).…”

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…Researchers have revealed the DNA-targeting specificities of CRISPR-based genome editing agents using a variety of approaches. These methods include ChIP-seq (Cencic et al, 2014;Kuscu et al, 2014;Wu et al, 2014;O'Geen et al, 2015), targeted analysis of genomic sites identified through computational predictions Hsu et al, 2013), in vitro high-throughput profiling methods (Pattanayak et al, 2013), whole-genome sequencing methods (Smith et al, 2014;Veres et al, 2014;Yang et al, 2014;Kim et al, 2015), the GUIDE-seq method , and the BLESS method (Crosetto et al, 2013;Ran et al, 2015). While detailed analyses of these methods are beyond the scope of this review, collectively they have revealed the presence of off-target activity among wild-type Cas9 homologs with certain sgRNAs and established that no simple algorithm or inspection process can accurately and comprehensively predict the offtarget substrates of a given Cas9:sgRNA complex (Tsai and Joung, 2016).…”