Off‐target assessment of CRISPR‐Cas9 guiding RNAs in human iPS and mouse ES cells

Abstract

Summary: The CRISPR-Cas9 system consists of a sitespecific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and~90 predicted off-target sites per gRNA. In a Cas9-based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human-induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near complementarity. genesis 53:225-236,

CRISPR, un système qui permet de corriger ou de modifier l’expression de gènes responsables de maladies héréditaires

“…Ce problème a cependant été atténué récemment [14]. En principe, 20 paires de bases (pb) sont plus que suffisantes pour être spécifiques d'un site cible unique dans le génome humain.…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…Further studies confirmed the absence of nonspecific activity by screening for cleavage activity at the most likely predicted off-target sites based on sequence homology to the guide RNA sequence [35,36,77]. Moreover, upon screening 624 and 717 potential off-target sites for 12 sgRNA in 293FT cells and iPSCs, respectively, a total of three sites were identified as being associated with two sgRNAs only [87]. On the other hand, Xu et al [138] showed clear off-target activity at seven out of 10 sites after targeting Intron 2 of the HBB gene, and reported a comparable level of off-target activity in 293FT cells and iPSCs.…”

“…In the context of this thesis, it is important to note that pluripotent stem cells adopt an open chromatin structure that differs from that of somatic cells [86], implying that iPSCs may have a higher propensity for off-target activity. Surprisingly, therefore, off-target Cas9 activity in iPSCs was reported to be low [87] or non-existent [88,89].…”

“…Generally, iPSCs exhibit a low off-target mutation load with Cas9WT as assessed by whole genome sequencing [88,89] or screening of the top predicted off-target sites [35,36,77,87].…”

“…However, it should be noted that Tan et al [87] limited their screen to an analysis of 90 offtarget sites for 12 gRNAs, whereas Smith et al [89] and Veres et al [88] used whole genome sequencing in their study, for only a limited number of sgRNAs, in iPSCs. This implies that the interplay of various factors determines Cas9 activity, including cell-type specific parameters that influence off-target activity and can potentially explain these apparently discordant findings, as discussed in Section 1.4. c) Modulation of the amount of sgRNA-Cas9 transfected: Off-target activity may be able to be reduced by limiting the amount of sgRNA-Cas9.…”