Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana

Li，Jing1,2; Wei，Qi1,2; Cheng，Yiqiu1,2; Kong，Dali1,2; Kong，Zhe1,2; Ke，Yongping1,2; Dang，Xiaofei1; Zhu，Jian Kang3; Shimada，Hiroaki4; Miki，Daisuke1

2024-12-01

Arabidopsis thaliana Cas12a Enhancer Gene targeting Genome engineering Sequential transformation

英语

Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

10.1186/s12870-024-05375-z

BMC Plant Biology

24

1

SCI

其他

2-s2.0-85198102497

Scopus

1471-2229

PLANT & ANIMAL SCIENCE

1.Shanghai Center for Plant Stress Biology,CAS Center for Excellence in Molecular Plant Sciences,Chinese Academy of Sciences,Shanghai,200032,China
2.University of Chinese Academy of Sciences,Beijing,100049,China
3.Institute of Advanced Biotechnology and School of Medicine,Southern University of Science and Technology,Shenzhen,518055,China
4.Department of Biological Science and Technology,Tokyo University of Science,Katsushika,Tokyo,125-8585,Japan

Li，Jing,Wei，Qi,Cheng，Yiqiu,et al. Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana. 2024-12-01.