Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice

Abstract

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR-Cas9-VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.

Keywords: CRISPR-Cas9; VQR; efficiency; genome editing; rice.

Figure 1

Modified single guide RNAs (sgRNAs) increased the efficiency of the clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9) system. (a) Schematic representation of modified sgRNAs. The replaced or introduced nucleotides are highlighted in red. These mutations abolish a potential transcription pause site and add a duplex extension for sgRNA stability. (b) The architecture of vectors in the CRISPR‐Cas9 system. The Cas9 protein attached to the nuclear localization signal (NLS) is driven by the 2x35S promoter. Both unmodified and modified sgRNAs are expressed by the U3 promoter. (c) The mutation rates at MOC3 and GW2. The modified sgRNA and unmodified sgRNA are represented in dark blue and light blue, respectively. (d) The proportion of mutants (biallelic or chimeric mutants) of moc3 and gw2. By modifying sgRNAs, the proportion of mutants increased approximately twofold or fivefold at MOC3 and GW2, respectively. (e) The proportion of moc3 gw2 double mutants. Modified sgRNAs dramatically increased (approximately 13‐fold) the proportion of double mutants.

Figure 2

Efficiency of the CRISPR‐Cas9‐VQR system is promoted by modified sgRNAs. (a) The architecture of vectors in the CRISPR‐Cas9‐VQR system. The VQR variants and sgRNAs are expressed by the 2x35S and U3 promoters, respectively. The binary vectors are constructed using one of the two sgRNAs. (b) The proportions of single mutations and double mutations. A, T and G, respectively, indicate the base following the NGA PAM. The unmodified sgRNA and modified sgRNA are represented in light blue and dark blue, respectively.

Figure 3

Efficiency of the CRISPR‐Cas9‐VQR system is further increased by the UBI1 and ACT1 promoters. (a) The architecture of vectors with different promoters. The promoters of VQR variants are replaced by UBI1 or ACT1. The modified sgRNAs are expressed with U3 promoters. (b) The mutation rate of the CRISPR‐Cas9‐VQR system using different promoters. The efficiency of the CRISPR‐Cas9‐VQR system is greatly increased by using strong promoters and modified sgRNAs. The system with the UBI1 promoter increases the mutation rate by an average of approximately fourfold, whereas that with ACT1 increases the mutation rate by an average of approximately sixfold. (c) The proportion of double and triple mutations using different systems. The proportions of double or triple mutations are dramatically increased. The triple mutations are undetected in the original system.