Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

• Published in: Nature communications Vol. 8; no. 1; p. 14261
• Main Authors: Liang, Zhen, Chen, Kunling, Li, Tingdong, Zhang, Yi, Wang, Yanpeng, Zhao, Qian, Liu, Jinxing, Zhang, Huawei, Liu, Cuimin, Ran, Yidong, Gao, Caixia
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.01.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 38/109; 38/22; 38/70; 38/77; 42; 42/41; 631/208/2491/2046; 631/337/4041/3196; 631/61/447/2311; CRISPR; CRISPR-Cas Systems; DNA, Plant - genetics; DNA, Plant - metabolism; Embryos; Gene Editing; Genetic testing; Genetics; Genomes; Humanities and Social Sciences; multidisciplinary; Mutagenesis; Mutation; Plant Breeding; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; Science; Science (multidisciplinary); Triticum - genetics; Triticum - metabolism; Wheat; Beijing China; China
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms14261

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized. Protocols for crop genome editing would ideally be quick, efficient and specific while avoiding integration of transgenes into the genome of edited plants. Here, Liang et al . show that CRISPR/Cas9 ribonucleoproteins can be used to generate genome edited wheat plants in as little as nine weeks.