Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato
Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato
ABSTRACT Genome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared to error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately 3-fold compared to 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 as well as sexually reproducing plants.
Sivankalyani Velu、Kim Eun-Jung、Vu Tien Van、Kim Jihae、Kang Yang Jae、Park Minwoo、Kim Jae-Yean、Sung Yeon Woo、Tran Mil Thi、Doan Duong Thi Hai
Division of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National UniversityDivision of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National UniversityDivision of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University||National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics InstituteDivision of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National UniversityDivision of Life Science, Gyeongsang National UniversityHyundai Seed Co., LTD.Division of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University||Division of Life Science, Gyeongsang National UniversityDivision of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National UniversityDivision of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National UniversityDivision of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University
农业科学技术发展生物科学研究方法、生物科学研究技术植物学
homology-directed repair (HDR)gene targetingCRISPR/Cpf1allele replacementMulti-replicon
Sivankalyani Velu,Kim Eun-Jung,Vu Tien Van,Kim Jihae,Kang Yang Jae,Park Minwoo,Kim Jae-Yean,Sung Yeon Woo,Tran Mil Thi,Doan Duong Thi Hai.Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato[EB/OL].(2025-03-28)[2025-08-02].https://www.biorxiv.org/content/10.1101/521419.点此复制
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