The CRISPR/Cas9 system has been widely used in plants to introduce genome modifications and is paving the way for precision crop trait improvement. Normally, CRISPR/Cas9 plasmids are delivered into plant cells by Agrobacterium tumefaciens mediated T-DNA transfer or biolistic bombardment, become expressed, cleave target sites, and produce mutations. During this process, there is a high possibility that the CRISPR/Cas9 plasmids may be integrated into plant genome, which increases the chance of producing unwanted genetic changes. Consequently, there are now substantial international efforts to optimize CRISPR/Cas9 mediated genome editing with the aim to avoid transgene integration and off-target mutations.
Recently, a research team led by Prof. GAO Caixia in the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences developed a DNA-free genome editing method in hexaploid wheat using the CRISPR/Cas9 ribonucleoproteins (RNPs).
In this study, the researchers delivered the CRISPR/Cas9 RNPs into wheat immature embryos by particle bombardment and succeeded in editing two different genes (TaGW2 and TaGASR7) in two varietal backgrounds (Kenong 199 and YZ814). In the model variety (Kenong 199), the mutation production efficiency (MPE) was highly as 4.4%. The MPE for the alternative bread wheat variety YZ184 was lower than for Kenong 199, indicating that the efficiency of CRISPR/Cas9 RNP-based genome editing may be genotype-dependent.
The researchers also found that the RNPs based method has an outstanding specificity when compared the methods based on CRISPR/Cas9 plasmids. Off-target mutations were drastically decreased in the recipient wheat cells, and absent in the recovered mutant population.
As the mutant plants edited using CRISPR/Cas9 RNPs do not have integrated transgenes, their application in practical breeding and their commercialization should be more publicly acceptable, thus accelerating precision crop improvement.
This work entitled “Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes” was published in Nature Communications
This research was supported by the National Natural Science Foundation of China, the Chinese Academy of Sciences, the Ministry of Science and Technology and the Ministry of Agriculture of China.
Dr. GAO Caixia