Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention

Abstract

Key messageWe present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations.AbstractSpecificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.Electronic supplementary materialThe online version of this article (10.1007/s11103-018-0709-x) contains supplementary material, which is available to authorized users.

A modular cloning toolkit for genome editing in plants

“…4). The tRNA-sgRNA system, originally described by Xie et al (2015) in rice, was later successfully applied in wheat [35] and Arabidopsis [36].…”

“…The tRNA-gRNA system for Cas9 multiplexing has proven to work in monocots [6,35] as well as in dicots [36]. In dicots, it was shown that fusing tRNAs with the optimised sgRNA backbone [39] increased editing efficiencies [36]. In monocots however, only the classic sgRNA backbone [40] was so far used in tRNA-sgRNA arrays.…”

An inducible genome editing system for plants

“…3; Supplementary table 1), indicating that sgRNA against PLT2 was disassociated from tRNA processing and guiding Cas9p to cleave PLT2 . It has recently been reported that efficient genome editing could be achieved by fusing tRNA to a mutant sgRNA scaffold but not the wild type sgRNA scaffold in Arabidopsis 36 . However, in our hands wild type sgRNA scaffold and tRNA fusion worked well.…”

CRISPR/Cas in Arabidopsis: overcoming challenges to accelerate improvements in crop photosynthetic efficiencies

“…In brief, sequence specificity is achieved by a short region (19–22 nt) in the gRNA that is complementary to the host target sequence and next to a 3‐ to 6‐nt protospacer adjacent motif (PAM) sequence (Table ; Jinek et al ). Off‐target mutations can occur at undesired sites that have mismatches distal to the PAM (Zhang et al ). However, several bioinformatic tools are now available to predict off‐target activity based on the gRNA(s) and Cas used, which can subsequently be screened for during analysis (reviewed in Zischewski et al ).…”