Development of PAM-flexible multiplex genome editors for enhancing cold-tolerance in indica rice
Jiantao Tan,
Kangli Sun,
Qian Qian,
Nan Chai,
Xin Peng,
Mengyuan Shen,
Degui Zhou,
Yao-Guang Liu,
Qinlong Zhu,
Qi Liu
Affiliations
Jiantao Tan
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China; Corresponding authors.
Kangli Sun
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China
Qian Qian
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China
Nan Chai
Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, South China Agricultural University, Guangzhou 510642, Guangdong, China
Xin Peng
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China
Mengyuan Shen
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China
Degui Zhou
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China
Yao-Guang Liu
Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, South China Agricultural University, Guangzhou 510642, Guangdong, China
Qinlong Zhu
Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, South China Agricultural University, Guangzhou 510642, Guangdong, China; Corresponding authors.
Qi Liu
Rice Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Genetics and Breeding of High-Quality Rice in Southern China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Rice Science and Technology, Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China; Corresponding authors.
The canonical CRISPR/Cas editors are constrained by the requirement for specific PAMs, which substantially limits their editable target range. Rice (particularly indica rice) is sensitive to low temperature, which impacts the yield and restricts the geographic distribution of rice. In this study, we developed PAM-flexible multiplex genome editing tools based on SpG (recognising NGN-PAMs) and SpRY (recognising NNN-PAMs) variants. We then tested the feasibility of using a sweet potato leaf curl virus (SPLCV) replicon-based expression vector and single-stranded DNA-binding domain (DBD) to improve the editing efficiency of these PAM-flexible editors. Furthermore, we used SpG-mediated multiplex genome editing to achieve comprehensive improvement in cold tolerance in indica rice by editing WRKY transcription factors OsWRKY53 and OsWRKY63, to generate high cold-resistant indica rice lines. We concluded that these PAM-flexible multiplex genome editors are powerful tools for multi-gene editing for crop genetic improvement.
