CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Abstract

CRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA.The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

An Analysis of gRNA Sequence Dependent Cleavage Highlights the Importance of Genomic Context on CRISPR-Cas Activity

“…We began by compiling data as discussed in the Methods Section, and illustrated in Figure 2a. 3–11,16,18–26 The datasets have varied distributions of cutting/cleavage activity, from binary distributions (gRNAs that either cut or do not cut) to skewed or normal distributions, suggesting significant experimental and context dependent differences in gRNA dependent activity (Figure 2b, data compiled in Supplementary File 1). Despite these differences, most datasets accurately capture the reported four nucleotide PAM preference of Cas9, highlighting that Cas9 specific features should correlate across contexts (Supplemental Figure S2).…”

“…17 Another example is the inhibitory search space (transient binding to “non-target sites”), part of the genomic context, which we have recently reported. 16 We demonstrated that the efficiency with which Cas9 cleaves a target site is decreased by the addition of inhibitory “non-target” sequences which are transiently interrogated by the Cas9/gRNA complex but not cleaved. 16 In the present study we sought to better understand the impact of context on gRNA activity and toward this goal we report a retrospective analysis of 39 gRNA library datasets from different species, with several Cas9 variants, using both endogenous and exogenous target sites, and in different experimental systems.…”

“…Broadly defined, “context” includes all variables outside of the gRNA/Cas9 complex that can impact activity, with the “genomic context” more specifically pertaining to the DNA sequences outside of the gRNA. 16 While significant work has been done to characterize the factors impacting Cas9 activity in vitro , there are limitations when comparing this to in vivo activity, particularly with respect to genomic context. The amount of competitive Cas9 binding sites 16 , target site accessibility, and other contextual in vivo factors are not easily replicated in vitro.…”

“…16 In the present study we sought to better understand the impact of context on gRNA activity and toward this goal we report a retrospective analysis of 39 gRNA library datasets from different species, with several Cas9 variants, using both endogenous and exogenous target sites, and in different experimental systems. 3–11,16,18–26 In this analysis we confirm the importance of context as a key factor driving gRNA dependent activity in vivo .…”

Anticipating and Identifying Collateral Damage in Genome Editing

“…In addition, noncanonical single guide RNA (sgRNA), Cas9 variants selected for enhanced specificity [73], and nickases [74] were used in initial strategies to achieve accurate interventions, in many cases to the detriment of efficiency. An alternative approach aims to focus activity on the desired target by increasing protospacer adjacent motif (PAM) selectivity, thereby diminishing activity at some other nontarget sites that harbor alternative PAM variants [75]. In addition, temporally controlling its activity by using ribonucleoprotein or a ubiquitin-proteasome degradation signal could help to restrict extensive DNA cutting [76].…”