PT  - JOURNAL ARTICLE
AU  - Tien Van Vu
AU  - Velu Sivankalyani
AU  - Eun-Jung Kim
AU  - Mil Thi Tran
AU  - Jihae Kim
AU  - Yeon Woo Sung
AU  - Duong Thi Hai Doan
AU  - Jae-Yean Kim
TI  - Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato
AID  - 10.1101/521419
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 521419
4099  - http://biorxiv.org/content/early/2019/02/28/521419.short
4100  - http://biorxiv.org/content/early/2019/02/28/521419.full
AB  - Genome editing via homology-directed repair (HDR) pathway in somatic plant cells was very inefficient compared to illegitimate repair by non-homologous end joining (NHEJ). Here, compared to a Cas9-based replicon system, we enhanced approximately 3-fold in the HDR-based genome editing efficiency via transient geminiviral replicon system equipping with CRISPR/LbCpf1 in tomato and obtained replicon-free, but with stable HDR alleles. Efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature or light. A ten-day incubation at 31 °C under light/dark cycles after Agrobacterium-mediated transformation performed the best among conditions tested. Further, we developed multi-replicon system which is a novel tool to introduce effector components required for the increase of HDR efficiency. Even if it is still challenging, we also showed a feasibility of HDR-based genome editing without genomic integration of antibiotic marker or any phenotypic selection. Our work may pave a way for transgene-free rewriting of alleles of interest in asexually as well as sexually reproducing plants.