An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

The CRISPR/Cas9 system is a revolutionary genome editing technique and has been widely used in numerous plants. For plants (e.g. citrus) with very low transformation efficiency, how to optimize gene editing efficiency and induce large-fragment deletion has been the focus of research. Here, we report that CRISPR/Cas9 induces efficient deletion of 16-673 bp fragments in the genome of Fortunella hindsii. The ability of two binary vectors, pK7WG2D and pMDC32, to introduce specific mutations into the genome of F. hindsii was evaluated. Double single guide RNAs (sgRNAs) were designed to achieve precise editing of two sites of a gene and deletion of fragments between the two sites. The construction of vectors based on Golden Gate assembly and Gateway recombination cloning is simple and efficient. pK7WG2D is more suitable for F. hindsii genome editing than the pMDC32 vector. Editing efficiency using the pK7WG2D vector reached 66.7%. Allele mutation frequency was 7.14-100%. Plants with 100% allele mutations accounted for 39.4% (13 100% allele mutation plants/33 mutants). The proportion of mutant plants with fragment deletion induced by this editing system was as high as 52.6% (10 fragment-deletion mutants/19 FhNZZ mutants). Altogether, these data suggest that our CRISPR/Cas9 platform is capable of targeted genome editing in citrus and has broad application in research on the citrus functional genome and citrus molecular breeding.