Many genetic and breeding studies have shown that single nucleotide changes can generate elite trait variations in crop plants. However, current methods of generating and identifying point mutations (e.g., Targeting Induced Local Lesions in Genomes, TILLING) are time-consuming and often detect a limited repertoire of point mutations.
Although the CRISPR/Cas9 system has been used to engineer point mutations in plants (e.g., rice), the efficiency of such practice is still low at present.
Thus, there is a pressing need for developing the genome engineering approaches that can introduce point mutations to targeted genomic sites in a highly efficient and precise manner.
Recently, a research team led by Prof. GAO Caixia from the Institute of Genetics and Developmental Biology of Chinese Academy of Sciences reported the optimization of an efficient and precise base editing approach (designated nCas9-PBE) for creating single nucleotide mutations in three major cereal crops including wheat, rice and maize.
In their study, the researchers successfully achieved cytosine to thymine substitutions in wheat, rice and maize using nCas9-PBE, whose main components include a Cas9 nickase-cytidine deaminase-uracil DNA glycosylase inhibitor fusion protein and a single guide RNA specific for the targeted genomic site.
Their results showed that nCas9-PBE changed C to T with the deamination window typically spanning from position 3 to 9 within the protospacer.
Their approach edited single C to T substitutions with the frequency varying from 0.39% to 7.07%. The frequency of editing multiple Cs (2 to 5) was even higher, ranging from 0.31% to 12.48%.
Furthermore, stable mutant plants carrying C to T mutations in OsCDC48, TaLOX2 or ZmCENH3 genes were generated, with the mutant production efficiency approaching 43.48%.
Clearly, nCas9-PBE is superior over other point mutation creation technologies currently available with respect to target specificity and efficiency.
"We believe that nCas9-PBE is an appealing new tool for producing the point mutations valuable in the precision breeding of wheat, rice and maize. This approach is also applicable for other crop plants, thus helping to increase the overall efficacy of crop improvement through genome engineering”, said Dr. GAO.
The research was supported by National Key Research and Development Program of China, National Program on Transgenic Plant Breeding, Chinese Academy of Sciences, and National Natural Science Foundation of China.
