Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Abstract

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBEC1 and PmCDA1 cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDA1, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

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).…”

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…Compared with SpCas9, xCas9 shows higher DNA specificity with considerably lower off‐target activity at all NGG PAMs on a genome‐wide scale, and the minimal off‐target activity at genomic regions with non‐NGG target sites . xCas9 has the ability to target canonical NGG PAMs while Cas9‐NG is a preferred enzyme to recognize relaxed PAMs for plant GE . The crystal structure of Cas9‐NG reveals that the newly added non‐base‐specific interactions can compensate the deficiency of base‐specific recognition with the third nucleotide base, which enable the recognition of NG PAM.…”

Expanding the base editing scope to GA and relaxed NG PAM sites by improved xCas9 system

“…The previously reported xCas9-CBEs were not efficient at NG PAM sites in rice regenerated plants, with the exception of canonical NGG PAM sites (Hua et al, 2019;Li et al, 2019;Ren et al, 2019;Zhong et al, 2019). Given the good performance of xCas9n-epBE at editing sites with GA PAM, we envisioned that xCas9n-epBE could also work on NG PAM sites in rice.…”

“…xCas9 showed broader PAM recognition including GAA and GAT than Cas9-NG and has been employed in base editors in human cells (Hu et al, 2018). However, no detected sites with these PAMs were edited by reported xCas9 involved cytosine base editors (xCas9-CBEs) in rice (Hua et al, 2019;Li et al, 2019;Ren et al, 2019;Zhong et al, 2019). In this study, we generated an efficient xCas9-CBE to achieve C-to-T mutation at GAA, GAT and even GAC, GAG PAM sites in rice, expanding the targeting scope and providing a reference for other plants and animals.…”

“…We first analyzed all reports on xCas9-CBEs and found that tRNA-sgRNA system was never used. Since xCas9 is a high-fidelity SpCas9 variant (Zhong et al, 2019), it may require perfectly matched 20nucleotide (nt) target like other high-fidelity Cas9 variants (Zhang et al, 2017). Studies showed that tRNA-sgRNA system can produce precise 20-nt sequence completely complementary to the targets by the cellular enzymes RNase P and RNase Z (Figure 1a).…”