CRISPR/Cas9 effectively generate chromosome structural variations in rice protoplasts

Chromosome structural variations (SVs), such as deletion, duplication, inversion, and translocation, are important contributors to genetic diversification and crop improvement. Using genome editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9), desired SVs involving large DNA fragments have been created in rice (Oryza sativa L.), maize (Zea mays L.), and Arabidopsis (Arabidopsis thaliana L.). However, it is still uncertain whether the size of DNA fragment involved could be a prohibiting factor to generate Cas9-mediated SVs. In this study, we constructed five CRISPR/Cas9 vectors, each expressing two single-guide RNAs (sgRNAs), to cut two sites spacing at 0.5, 5, 10, 20, and 30 Mb on rice chromosome 4 (Chr4), respectively. Meanwhile, another CRISPR/Cas9 vector cutting two sites, one on Chr4 and the other on Chr1, was also constructed for creation of chromosomal translocation between Chr1 and Chr4. These vectors were transfected into rice protoplasts by polyethylene glycol–mediated transformation. Specific primers were designed to detect desired SV events. The results showed that all designed SVs could be effectively generated by CRISPR/Cas9 in rice protoplasts. This study suggested that the size of DNA fragment involved is unlikely a prohibiting factor for creation of desired SV events.