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

Plant Molecular Biology
Qiang ZhangQi-Jun Chen

Abstract

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. 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 h...Continue Reading

References

Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 5, 2013·Science·Prashant MaliGeorge M Church
Jun 25, 2013·Nature Biotechnology·Yanfang FuJeffry D Sander
Jul 23, 2013·Nature Biotechnology·Patrick D HsuFeng Zhang
Sep 12, 2013·Nature Biotechnology·Dana Carroll
Feb 20, 2014·Proceedings of the National Academy of Sciences of the United States of America·Zhengyan FengJian-Kang Zhu
May 20, 2014·The Plant Journal : for Cell and Molecular Biology·Friedrich FauserHolger Puchta
Nov 30, 2014·BMC Plant Biology·Hui-Li XingQi-Jun Chen
Feb 28, 2015·Quantitative Biology·Xuebing WuPhillip A Sharp
Mar 4, 2015·Proceedings of the National Academy of Sciences of the United States of America·Kabin XieYinong Yang
Oct 20, 2015·Nature Biotechnology·Je Wook WooJin-Soo Kim
Oct 31, 2015·Current Opinion in Chemical Biology·Henriette O'GeenDavid J Segal
Nov 6, 2015·Genome Biology·Dana Carroll, R Alta Charo
Nov 19, 2015·The Plant Journal : for Cell and Molecular Biology·Jeannette SteinertHolger Puchta
Dec 3, 2015·Science·Ian M SlaymakerFeng Zhang
Dec 17, 2015·Genome Biology·Ying DangHaoquan Wu
Jan 19, 2016·Nature Biotechnology·John G DoenchDavid E Root
Apr 19, 2016·Nature Reviews. Genetics·Shengdar Q Tsai, J Keith Joung
Aug 6, 2016·Molecular Cell·Josh TyckoPatrick D Hsu
Nov 17, 2016·Nature Communications·Sergei SvitashevA Mark Cigan
Dec 3, 2016·The Plant Genome·Jeffrey D WoltCarolyn J Lawrence-Dill
Dec 13, 2016·The Plant Journal : for Cell and Molecular Biology·Javier Gil-HumanesDaniel F Voytas
May 20, 2017·The Plant Cell·Tomáš ČermákDaniel F Voytas
Aug 2, 2017·Nature Plants·Kangquan YinJin-Long Qiu
Sep 21, 2017·Nature·Janice S ChenJennifer A Doudna
Oct 8, 2017·Genome Biology·Péter István KulcsárErvin Welker

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Citations

Jan 16, 2019·Plant Molecular Biology·Keunsub LeeKan Wang
Jul 19, 2019·Journal of Cellular Physiology·Niaz AhmadBaohong Zhang
Jul 17, 2019·Nature Plants·Yingxiao ZhangYiping Qi
Apr 10, 2020·Plant Biotechnology Journal·Debin ZhangFang Ding
Apr 16, 2020·G3 : Genes - Genomes - Genetics·Renyu LiCristian H Danna
Aug 28, 2019·International Journal of Molecular Sciences·Wenjie XuShuifang Zhu
Aug 28, 2020·Frontiers in Plant Science·Kayla Beam, José Trinidad Ascencio-Ibáñez
Jan 12, 2020·Journal of Applied Genetics·Luiza Chojnacka-Puchta, Dorota Sawicka
May 15, 2020·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·Sukumar BiswasJianxin Shi
Sep 27, 2018·Current Genetics·Neil R AdamesJean Peccoud
Apr 25, 2020·BMC Plant Biology·Florian HahnVladimir Nekrasov
Nov 15, 2018·Plant Cell Reports·Florian Hahn, Vladimir Nekrasov
Aug 2, 2019·International Journal of Molecular Sciences·Zahra HajiahmadiQiang Zhuge
Nov 18, 2020·Journal of Plant Physiology·Jie LiuJianbing Yan
Nov 17, 2020·Frontiers in Microbiology·Yongsen CaoFangfang Li
Feb 2, 2021·Science China. Life Sciences·Zhen LiangYincen Wu
Mar 2, 2021·Transgenic Research·Teng-Kuei Huang, Holger Puchta
Apr 15, 2018·Plant Physiology and Biochemistry : PPB·Ulhas Sopanrao KadamPenna Suprasanna
Aug 4, 2021·Trends in Plant Science·Md Mahmudul HassanXiaohan Yang
Aug 28, 2021·International Journal of Molecular Sciences·Stanton B Gelvin
Sep 25, 2021·ACS Synthetic Biology·Cen LiSong Liu
Dec 12, 2021·Plant Physiology·Jin-Lei LiuXiu-Fen Song

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Methods Mentioned

BETA
PCR
transgenic
Seq

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