Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato.

Autor: Vu TV; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea.; National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Bac Tu Liem, Vietnam., Sivankalyani V; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea., Kim EJ; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea., Doan DTH; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea., Tran MT; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea., Kim J; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea., Sung YW; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea., Park M; Hyundai Seed Co., LTD., Yeoju, Korea., Kang YJ; Division of Life Science, Gyeongsang National University, Jinju, Korea., Kim JY; Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea.; Division of Life Science, Gyeongsang National University, Jinju, Korea.
Jazyk: angličtina
Zdroj: Plant biotechnology journal [Plant Biotechnol J] 2020 Oct; Vol. 18 (10), pp. 2133-2143. Date of Electronic Publication: 2020 Apr 01.
DOI: 10.1111/pbi.13373
Abstrakt: Genome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared with error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately threefold compared with a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31 °C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mm NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually and sexually reproducing plants.
(© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)
Databáze: MEDLINE
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