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. Core Ideas Deletion, duplication, and inversion events were generated in rice protoplasts by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9). Translocation events were generated in rice protoplasts by CRISPR/Cas9. The size of DNA fragment involved is unlikely a limiting factor for creation of desired structural variation events. Plain Language Summary This study focuses on using CRISPR/Cas9 genome editing tools to create specific structural variations (SVs) in rice chromosomes. SVs are changes in the structure of chromosomes, like deletions, duplications, inversions, and translocations, which can contribute to genetic diversity and crop improvement. This study aimed to determine if the size of the DNA fragment involved in the SV creation using CRISPR/Cas9 is a limiting factor. Cas9 vectors were designed and constructed to target different distances on the same chromosome and even between different chromosomes. When these vectors were transfected into rice protoplasts, the desired SVs were readily detected, suggesting that the size of the DNA fragment inv