Directed evolution of CRISPR-Cas9 to increase its specificity

Abstract

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we have used a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without sacrificing on-target activities in human cells. Main textThe determination of the Cas9 crystal structure 1 enabled scientists to rationally design mutant Cas9 proteins (enhanced SpCas9 (eSpCas9) and Cas9-High Fidelity (Cas9-HF)) with higher specificities than wild-type Cas9 (WT-Cas9) 2, 3 . Their design was based on the hypothesis that weakening non-specific interactions between a Cas9-RNA complex and its substrate DNA would reduce both on-target and off-target activities alike. Since on-target activity is generally much higher than off-target activity, these mutant Cas9 variants would show higher specificities than WT while retaining on-target activities. However, many groups have since reported that both eSpCas9 and Cas9-HF were poorly active at many targets tested 4-6 , calling for alternative approaches to improve Cas9 specificity.We reasoned that directed evolution of Cas9 in E. coli could lead to Cas9 variants with high specificity without sacrificing on-target activities. The system we used consists of E. coli strain BW25141 and a plasmid containing the lethal ccdB gene 7-9 and the Cas9 target sequence: The disruption of the ccdB gene by Cas9-mediated plasmid DNA cleavage is essential for cell survival, creating a positive selection pressure. In addition, a Cas9 off-target sequence that differs from the on-target sequence by a few mismatches is introduced in the E. coli genomic DNA: Double strand breaks (DSBs) in E. coli genomic DNA lead to cell death. We combined such negative selection pressure with ccdB plasmid-based positive selection pressure to develop 'Sniper-screen', which selects for Cas9 variants with increased specificities. Note that Cas9 variants with poor on-target activities or poor specificities cannot survive in this selection system.We first inserted a 500-bp PCR product containing an EMX1 fragment into the genomic DNA of the BW25141 strain using a protocol involving transposase 10 (Figure 1a and

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

“…Wang et al, 2019) seems not as effective as originally in human cells (Hu et al, 2018). It has been suggested that some Cas9 variants including xCas9 3.7 have much greater DNA specificity than the wild-type Cas9 (Hu et al, 2018;Lee et al, 2018;Rees et al, 2017). The transcribed gRNA used here has an extra A at the 5'-end of 20-nt guide sequence, which may affect the pairing of gRNA with protospacer and consequently affect the editing efficiency of xCas9 3.7 ( Figure S2).…”

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

“…Following binding, Cas9-RNA activates its two nuclease domains (HNH for target strand and RuvC for non-target strand) and cleaves the DNA, producing a double-strand break [3][4][5][6][7][8][9] . Several Cas9 variants have been engineered (Engineered Cas9; EngCas9) to improve cleavage specificity [10][11][12][13][14][15][16][17] . Stable binding by Cas9s requires as few as 9-10 bp between the PAM-proximal end of the protospacer and the gRNA, but cle avage re quire s more bps (~16 for WT Cas9 and ~17-18 for EngCas9s) 5,7,18 .…”

“…1a) that the DNA in Cas9-RNA-DNA undergoes internal unwinding-rewinding dynamics and the unwound state is a critical checkpoint required for cleavage. The intrinsic rate of cleavage (kc, int) has been used to refer to the rate of cleavage from the unwound state 15,18 . The PAM-distal mismatches reduce the lifetime and extent of the unwound state, thereby delaying or preventing cleavage 18 .…”

Editing the Reactive Genome: Towards a Postgenomic Ethics of Germline Editing

“…This method has already been used to optimise the performance of CRISPR-Cas9, the main tool currently used in genome editing. 29 Clearly, this path forward relies on more than just the research use of GGE. We have here a general research context that is separated from clinical uses of GGE and that is safe, as no humans are exposed to harm (directed evolution, for instance, is a microbe-based research system).…”