Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing inSetaria viridis
文献类型： 外文期刊
作者： Weiss, Trevor 1 ; Wang, Chunfang 1 ; Kang, Xiaojun 1 ; Zhao, Hui 5 ; Elena Gamo, Maria 2 ; Starker, Colby G. 2 ; Crisp 1 ;
作者机构： 1.Univ Minnesota, Dept Plant & Microbial Biol, Coll Biol Sci, Minneapolis, MN 55108 USA
2.Univ Minnesota, Ctr Plant Precis Genom, Minneapolis, MN 55108 USA
3.Univ Minnesota, Microbial & Plant Genom Inst, Minneapolis, MN 55108 USA
4.Univ Minnesota, Ctr Genome Engn, Minneapolis, MN 55108 USA
5.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Haikou 571101, Hainan, Peoples R China
6.Chinese Acad Trop Agr Sci, Hainan Key Lab Biosafety Monitoring & Mol Breedin, Haikou 571101, Hainan, Peoples R China
7.Univ Minnesota, Dept Genet Cell Biol & Dev, Minneapolis, MN 55108 USA
8.Univ Queensland, Sch Agr & Food Sci, Brisbane, Qld 4072, Australia
关键词： Cas9; CRISPR; multiplexed genome editing; NHEJ and MMEJ DNA repair; Setaria viridis; Trex2exonuclease; technical advance
期刊名称：PLANT JOURNAL （ 影响因子：6.417； 五年影响因子：7.627 ）
ISSN： 0960-7412
年卷期： 2020 年 104 卷 3 期
页码：
收录情况： SCI
摘要： In recent years,Setaria viridishas been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources forS. viridisresearch, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system inS. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease,Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robustAgrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes,svDrm1aandsvDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource forS. viridisin a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.
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