Repositorium
Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention
389
Journal Article / 2018
Zhang, Qiang; Xing, Hui-Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Yang, Fang; Wang, Xue-Chen; Chen, Qi-Jun
Key message We 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. 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 wildtype 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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
896 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 CPC |
No information SDN1 |
Basic research Basic research |
897 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 ETC2 |
No information SDN1 |
Basic research Basic research |
898 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 TRY |
No information SDN1 |
Basic research Basic research |
899 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 AT5G50230 |
No information SDN1 |
Basic research Basic research |
900 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 BRI1 |
No information SDN1 |
Basic research Basic research |
901 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 HAB1 |
No information SDN1 |
Basic research Basic research |
902 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 ADH1 |
No information SDN1 |
Basic research Basic research |