Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

Abstract

Background: Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits.

Results: We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture.

Conclusions: We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

Keywords: Base editing; CRISPR/Cas9; High-fidelity Cas9 variants; Off-target effect; tRNA-sgRNA.

Fig. 1

Comparation of the editing efficiency of rBE and pBE. a Vector structure used for C-T substitution in both rAPOBEC1 base editing (rBE) and PmCDA1 base editing (pBE) system. tRNA-sgRNA architecture used for multiplex editing was also presented. Three or four targets were assembled to test the editing efficiency. b Comparation of C to T substitution efficiency edited by both SpCas9-rBE and SpCas9-pBE system at seven genomic targets guided by wild type sgRNA. For each construction, fifteen transgenic calli were selected independently for Sanger sequencing. Substitution efficiency was defined as the percentage of calli containing C to T conversion at any position in the editing targets

Fig. 2

Multiplex nucleotide editing using SpCas9 and sgRNA variants. a High-fidelity Cas9 variants with their specific mutation sites used in this study. The crystal structure of SpCas9 protein was download from the structure resource of NCBI (PDB ID: 6O0Y). b Schematic representation of modified sgRNA. The polyU mutation was marked with brown and the introduced nucleotides in the extend loop were highlighted in red. N20 with purple indicated targeted DNA. c Comparation of C to T editing efficiency of four SpCas9 variants directed by tRNA-sgRNA and tRNA-modified sgRNA in pBE system. Two independent transformations were carried out to repeat the editing efficiency. Substitution efficiency was defined as the percentage of calli containing C to T conversion at any position in the editing targets. Samples with significant increasement were marked out using the black line. d Comparation of random mutation efficiency of four SpCas9 variants directed by tRNA-sgRNA and tRNA-modified sgRNA at three targets. Two independent transformations were carried out to repeat the editing efficiency. Indel frequency was defined as the percentage of calli containing deletion or insertion in the editing targets. Samples with significant increasement were marked out using the black line

Fig. 3

Specificities of high-fidelity Cas9 variants. a Off-target activity detected at three potential off-target sites of ALS, CDC48 and NRT1.1B in the genomics in pBE system. The off-target efficiency was detected using deep sequencing described in method. b Off-target effects of four Cas9 variants detected with guide sequences containing pairs of mismatches at successive positions combined with tRNA-sgRNA and tRNA-modified sgRNA in pBE system. The mismatch sites were highlighted in red with the PAM highlighted in blue. c Off-target activity of SpCas9 and eSpCas9(1.1) detected on two potential off-target sites of GW8-T2 in the genomics using deep sequencing during random mutation