Engineered CRISPR-Cas9 nuclease with expanded targeting space

Abstract

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

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

“…nCas9(D10A)‐AID showed the highest editing frequency over 95% for targets with the canonical NGG PAM (Figure b and Table S6). Weak NAG PAM and non‐canonical NGA PAMs were also captured by nCas9(D10A)‐AID for base editing, which was consistent with previous reports (Hu et al, ; Nishimasu et al, ). The editing frequencies for targets with NAG or NGA PAMs suggest bias against a C in the +1 position of PAM sequence as previously suggested (Leenay et al, ).…”

“…The editing efficiencies of nCas9‐NG(D10A)‐AID with NG PAMs were 3‐ to 28‐fold higher than nCas9(D10A)‐AID and 1.5‐ to 4.2‐ fold higher than nxCas9 3.7(D10A)‐AID (Figure ). Editing events at targets with several NA PAMs were also observed, such as GAT, AAT, GAA, and CAT (Figure b and Table S6), which were consistent with the slight recognition of NA PAMs reported by its inventors (Nishimasu et al, ). Although application of xCas9 3.7 and Cas9‐NG expanded the targeting scope for base editing, the editing window and product purity were generally not changed (Figures S4‐S7 and Table S6).…”

Expanding the scope of CRISPR/Cas9‐mediated genome editing in plants using an xCas9 and Cas9‐NG hybrid

“…The engineered variant Cas9‐NG was reported to have higher editing efficiency than xCas9 at NG sites in human cells (Nishimasu et al ). We introduced the reported point mutations R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R (Nishimasu et al ) in plant codon‐optimized SpCas9 to generate Cas9‐NG using overlap PCR (Figure A, Table S1). In addition, we combined the xCas9 mutations with Cas9‐NG mutations (A262T/R324L/S409I/E408K/E543D/M694I/R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R) to generate the XNG‐Cas9 using overlap PCR (Figure A).…”

“…The target sequences of the most widely used Cas, SpCas9, are restricted to genomic sites containing an NGG protospacer adjacent motif (PAM), thus limiting the availability of suitable target sites and reducing the utility of SpCas9. Recently, the SpCas9 variants xCas9 and Cas9‐NG have been developed to recognize not only the canonical NGG PAM but also NG, GAA and GAT PAMs in human cells (Hu et al ; Nishimasu et al ). These variants have also been adapted to expand the scope of CRISPR/Cas9 genome editing in Arabidopsis and rice (Kang et al ; Wang et al ; Ge et al ; Hua et al , ; Wang et al ).…”

Impact of CRISPR interference on strain development in biotechnology

“…The requirement of a PAM sequence (e.g., NGG for SpdCas9) inherently limits the number of potential target sequences [157,158]. This may be overcome either by dCas9 enzyme engineered to recognize more PAM sequences [51,159] or by using other CRISPRi systems such as that based on ddCas12a [160,161].…”