xCas9 expands the scope of genome editing with reduced efficiency in rice

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

“…Even at the canonical NGG PAM sites, the editing efficiencies of xCas9 were very low (3.9%, 2.8%, and 2.9% at GGG, TGG, and AGG sites, respectively) (Figure C, Tables S5, S6). This low editing efficiency of xCas9 in tomato is consistent with results from studies in rice (Wang et al ).…”

“…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 ).…”

Engineered xCas9 and SpCas9‐NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants

“…Taken together, the results of this study expand the application of engineered Cas9 variants that recognize non-NGG PAMs in Arabidopsis in planta. Similar results have recently been reported in rice while this paper is being reviewed, in which xCas9 and SpCas9-NG variants successfully recognize non-canonical PAMs and induce mutations, with reduced efficiency than wild-type Cas9 on NGG PAMs (Hua et al, 2019;Ren et al, 2019;Wang et al, 2018;Zhong et al, 2019). We propose that this phenomenon is a consequence of the Agrobacterium-mediated transformation method used in the plants, especially in Arabidopsis, which is quite different from the transfection methods used for mammalian cell transformation, in which in vitrotranscribed Cas9-encoding mRNA and SgRNAs are coinjected (Nishimasu et al, 2018;Wang et al, 2018).…”

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

“…In this study, four Cas9 variants, VQR‐Cas9, VRER‐Cas9, xCas9 3.7, and Cas9‐NG, were used for base editing in C. glutamicum . Because xCas9 3.7 and Cas9‐NG were very recently reported and have only been applied in human cells and plants (Endo et al, ; J. Wang et al, ), their PAM specificity and efficiency were comprehensively evaluated in C. glutamicum . Although both nxCas9 3.7(D10A)‐AID and nCas9‐NG(D10A)‐AID displayed the ability to edit noncanonical PAM sites, nCas9‐NG(D10A)‐AID showed more stable editing efficiency in NG PAM sites, which provided 3.9‐fold more accessible sites for C. glutamicum gene inactivation than the original nCas9(D10A)‐AID.…”

“…Although both nxCas9 3.7(D10A)‐AID and nCas9‐NG(D10A)‐AID displayed the ability to edit noncanonical PAM sites, nCas9‐NG(D10A)‐AID showed more stable editing efficiency in NG PAM sites, which provided 3.9‐fold more accessible sites for C. glutamicum gene inactivation than the original nCas9(D10A)‐AID. The xCas9 3.7‐mediated base editing in C. glutamicum and rice (J. Wang et al, ) seems not as effective as originally in human cells (Hu et al, ). 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, ; Lee et al, ; Rees et al, ).…”