Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat
The great advances in exploiting the CRISPR/Cas9 system are paving the way for targeted genome engineering in plants. Genome editing by direct delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs) into plant cells reduces off-target mutations and avoids the integration of foreign DNA fragments, thus providing an efficient and accurate method for precision crop breeding. Here we describe an RNP-based genome editing protocol for wheat. The protocol covers the in vitro transcription of sgRNA, purification of Cas9 protein, biolistic delivery of CRISPR/Cas9 RNPs, and tissue culture procedures for regenerating testable seedlings.
Key wordsCRISPR/Cas9 Ribonucleoprotein Biolistic delivery Wheat
This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the National Transgenic Science and Technology Program (2016ZX08010-002), the Chinese Academy of Sciences (QYZDY-SSW-SMC030 and GJHZ1602), and the National Natural Science Foundation of China (31788103, 31420103912 and 31570369).