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

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.