Scientist from Viet Nam National Key Laboratory for Plant Cell Biotechnology and Gyeongsang National University, Republic of Korea, show important improvement of HDR in tomato in a recent paper published in bioRxiv. By using a de novo engineered geminiviral replicon system that expressed CRISPR/LbCpf1, as a DNA double stranded break induction tool, and high doses of a homologous donor template they achieved about three-fold increase of HDR efficiency compared to a similar CRISPR/Cas9 replicon system published by other group (Cermark et al., Genome Biology, 2015).
The HDR was shown by perfectly sequence exchanging between their introduced donor template and an endogenous ANT1 allele. Further they developed multi-replicon system which is a novel tool to introduce effector components required for the increase of HDR efficiency. More importantly, they demonstrate that the events carrying the HDR-edited alleles are all free of replicon even from the first generation.
They also succeeded to have a perfect HDR GE0 event to produce salt tolerant allele (N217D) using their system with a HKT1;2 gene donor template which contains neither antibiotic selection marker nor ANT1 color marker. It should be noted that the mutated nucleotide (A to G) of HKT1;2 is not accessible by any currently known base editor (BE) including xCas9-ABE, underlining the significance of HDR-based genome editing.
The work may pave a way for transgene-free rewriting of alleles of interest in asexually as well as sexually reproducing plants.
Article information
Homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato
Tien Van Vu, Velu Sivankalyani, Eun-Jung Kim, Mil Thi Tran, Jihae Kim, Yeon Woo Sung, Duong Thi Hai Doan, Jae-Yean Kim
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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).