Researchers Develop a Precise and Efficient Scarless Large DNA Fragments Editing Technology in Plants

Improving agronomic traits relies on the exploitation and manipulation of plant genetic variation, ranging from single nucleotide polymorphisms to large structural variants (SVs). In particular, SVs represent a major source of genetic diversity and play critical roles in genome evolution and the genetic control of agronomical traits in plants. However, manipulation of larger DNA fragments, such as large deletions, replacements, and inversions in the tens of kilobases, remains a significant challenge in plant genomes.

 

To tackle these challenges, a team led by Professor WANG Yanpeng from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences, in collaboration with the Wheat Genetics and Genomics Center of China Agricultural University, develop a precise and efficient scarless large DNA fragments technology in plants, which can efficiently facilitate precise deletion, replacement, and inversion of large DNA fragments in plants. This study was published in Nature Plants (https://doi.org/10.1038/s41477-024-01898-3).

 

This study used an optimized prime editor, ePPEplus, which contains a V223A mutation in the reverse transcriptase and the R221K and N394K mutations in nCas9 (H840A), with the dual epegRNAs to target complementary DNA strands with a PAM-in orientation. The ePPEplus-epegRNA complex recognizes the target sites adjacent to the large DNA fragment being edited and nick the PAM-containing DNA strands, respectively. The 3′ end of the nicking sites hybridize to the corresponding PBS of epegRNA and then reverse transcribe RT templates (RTTs), generating two 3′ DNA flaps at the respective target sites. Theoretically, as depicted in Figure, through the manipulating of these3′ DNA flaps, it is possible to achieve large DNA deletions, replacements, or inversions.

 

Here are the possible scenarios: (1) If each 3′ flap sequence is complementary to the region targeted by the other pegRNA, the two 3′ flaps will anneal to form double-stranded DNA, resulting in the excision of the original DNA strands (5′ flaps). The subsequent DNA repair process leads to the precise deletion of DNA fragments between the two pegRNAs. (2) In cases where the two 3′ flaps generated by the RTT are partially complementary to each other and also contain a desired insertion sequence, two 3′ flaps annealing, followed by DNA synthesis and excision of the 5′ flaps, results in a deletion and simultaneous insertion of the desired sequence between the two pegRNAs. (3) When each 3′ flap sequence is complementary to the inverted region targeted by the other pegRNA, each 3′ flap will anneal to its corresponding sequence. This annealing will serve as a primer to initiate DNA synthesis in the opposite orientation. Subsequently, the 5′ flaps will be excised, followed by ligation and DNA repair, which may result in an inversion. These three scenarios represent the genome-editing approach that we term dual prime editing (DualPE).

 

The results showed that DualPE efficiently facilitates precise deletion, replacement, and inversion of large DNA fragments in plants. DualPE enabled the production of specific genomic deletions ranging from ~500 bp to 2 Mb in wheat protoplasts and plants. DualPE was effective in directly replacing wheat genomic fragments of up to 258 kb with desired sequences in the absence of donor DNA. Additionally, DualPE allowed precise DNA inversions of up to 205.4 kb in wheat plants with efficiencies of up to 51.5%. DualPE also successfully edited large DNA fragments in dicotNicotiana benthamiana and tomato, with editing efficiencies of up to 72.7%. This study also developed the websever DualPE-Finder to facilitate dual-pegRNA design for large DNA fragment editing.

 

DualPE thus provides a precise and efficient approach for large DNA sequence and chromosomal engineering, expanding the availability of precision genome editing for crop improvement.

 

DualPE-mediated large DNA deletions, replacements, and inversions in plant (Image by IGDB)

 

 

Contact:

Prof. WANG Yanpeng

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Email: yanpengwang@genetics.ac.cn