Due to the simplicity and high-efficiency, the CRISPR/Cas9 has been almost applied in all the organisms for functional analysis and trait improvements. Currently, delivery of the CRISPR/Cas9 reagents into plant cells is still a major bottleneck for further applications.
Recently, researchers from Dr. GAO Caixia's group at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, developed protocols for DNA-free genome editing in hexaploid wheat by biolistic delivered CRISPR/Cas9 IVT or RNP.
CRISPR/Cas9 components can be delivered to cells in three forms: DNA expression plasmids (DNA), in vitro transcripts (IVTs) and preassembled ribonucleoprotein complexes (RNPs).
Conventional genome editing in plants delivered the CRISPR/Cas9 DNA into plant cells by Agrobacterium or biolistic transformation. However, the integrated CRISPR/Cas9 DNA can constitutively function and increase the off-target effect.
Also, cross or backcross is needed to segregate the CRISPR/Cas9 cassettes. Therefore, delivery methods for CRISPR/Cas9 IVTs or RNPs are urgently needed genome editing in plants.
The whole procedure of GAO's protocol includes preparation of IVTs and RNPs; validation by in vitro cleavage and transient protoplast assay; delivery by biolistic transformation; recovery of mutants and identification of mutants by pooling methods can be accomplished within 9 to 11 weeks.
Genome editing based on the CRISPR/Cas9 IVT or RNP can avoid foreign DNA integrated into host genome and reduce off-target effect. By using this protocol, foreign DNA-free genome-edited plants can be obtained in T0 generation.
Dr. GAO said: "We believe that this protocol is useful to improve the genome editing in plants and accelerate the process of precision breeding."
This research was supported by grants from the National Key Research and Development Program of China, the National Transgenic Science and Technology Program, the Chinese Academy of Sciences, and National Natural Science Foundation of China.
