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

Abstract

Specificity 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.

Reduced fire blight susceptibility in apple cultivars using a high‐efficiency CRISPR/Cas9‐FLP/FRT‐based gene editing system

“…However, its application is associated with off‐targeting, that is the generation of unwanted mutations in off‐target genomic sites. In Arabidopsis (Zhang et al ., ), barley (Lawrenson et al ., ), Brassica oleracea (Lawrenson et al ., ), rice (Tang et al ., ), apple (Charrier et al ., ) and pear (Charrier et al ., ), the occasionally off‐targeting has been reported to be a potential issue when CRISPR/Cas9 was applied, especially if the guide RNA shared significant similarity (complete homology or 1 mismatch) with OT sequences. On the contrary, in apple (Charrier et al ., ) and grape (Wang et al ., ) no editing activity was found by the analysis of OT regions showing three or four mismatches with the guide RNAs.…”

“…Following the generation of mutations at the targeted genomic site, the Cas9‐guide RNA complex is processed and degraded resulting in edited protoplasts free from exogenous editing machinery, some of which can be regenerated into a new plant. In Arabidopsis , lettuce, petunia, rice, tobacco and wheat, this methodology was successfully reported (Subburaj et al ., ; Woo et al ., ; Zhang et al ., ). Nevertheless, the strategy can neither be widely applied nor represent an alternative to the conventional Agrobacterium tumefaciens ( A. tumefaciens )‐mediated transformation as, to date, efficient protocols for protoplast regeneration are still not available for many plant species.…”

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

“…To minimize off-target effects, multiple high-fidelity Cas9 variants were developed (Kleinstiver et al, 2016;Slaymaker et al, 2016;Chen et al, 2017;Casini et al, 2018;Lee et al, 2018;Vakulskas et al, 2018). However, adoption of some of these highfidelity Cas9 variants into plants faced low editing efficiency while success in their use required precisely defined protospacer length of the single guide RNAs (sgRNAs) (Zhang et al, 2017) and high expression level of sgRNAs (Zhang et al, 2018).…”

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

“…Considering this possibility, we reasoned that enhancing pegRNA expression would improve the editing efficiency. We used two strategies to enhance pegRNA expression: doubling the number of pegRNA expression cassettes and using two promoter systems to drive pegRNA expression, together with tRNA, ribozyme, and Csy4 RNA processing systems [12,13] as appropriate (Fig. 1b).…”