Journal
PLANT JOURNAL
Volume 104, Issue 3, Pages 828-838Publisher
WILEY
DOI: 10.1111/tpj.14949
Keywords
Cas9; CRISPR; multiplexed genome editing; NHEJ and MMEJ DNA repair; Setaria viridis; Trex2exonuclease; technical advance
Categories
Funding
- College of Biological Sciences, University of Minnesota
- DOE [DE-SC0018277]
- key research and development project of Hainan [ZDYF2017017]
- NSF [IOS-1802848]
- University of Minnesota
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available