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

bioRxiv preprint first posted online Jan. 16, 2019; doi: http://dx.doi.org/10.1101/521419. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Authors
Tien Van Vu, Velu Sivankalyani, Eun-Jung Kim1, Mil Thi Tran1, Jihae Kim , Yeon Woo Sung, Duong Thi Hai Doan1, Jae-Yean Kim.
04-03-2019 HSW1057
12:00pm
PST
Categories
RNA Modification & Editing
Speaker
Abstract


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 o19 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.