Transposase-assisted target-site integration for efficient plant genome engineering

• Published in: Nature (London) Vol. 631; no. 8021; pp. 593 - 600
• Main Authors: Liu, Peng, Panda, Kaushik, Edwards, Seth A., Swanson, Ryan, Yi, Hochul, Pandesha, Pratheek, Hung, Yu-Hung, Klaas, Gerald, Ye, Xudong, Collins, Megan V., Renken, Kaili N., Gilbertson, Larry A., Veena, Veena, Hancock, C. Nathan, Slotkin, R. Keith
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18-07-2024; Nature Publishing Group
• Subjects: 42; 42/41; 45; 631/1647/1511; 631/208/2491/2046; 631/337/2569; 631/449/447/2311; 64; Amino acid sequence; Arabidopsis - genetics; Chromatin; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA Transposable Elements - genetics; Enhancer Elements, Genetic - genetics; Gene Editing - methods; Gene expression; Genetic Engineering - methods; Genome editing; Genome, Plant - genetics; Genomes; gRNA; Humanities and Social Sciences; Insertion; multidisciplinary; Mutagenesis, Insertional - genetics; Mutation; Nucleotide sequence; Open Reading Frames - genetics; Oryza - enzymology; Oryza - genetics; Parasites; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified - genetics; Proteins; Rice; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Science; Science (multidisciplinary); Soybeans; Transposase; Transposases - genetics; Transposases - metabolism; Transposition
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/s41586-024-07613-8

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops
1
,
2
. Often considered to be genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes
3
–
5
. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type
6
–
9
. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria
10
–
12
, we fused the rice
Pong
transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant
Arabidopsis
. We then translated this system into soybean—a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.
Fusion of rice Pong transposase to the Cas9 or Cas12a programmable nucleases provides sequence-specific targeted insertion of enhancer elements, an open reading frame and gene expression cassette into the genome of the model plant
Arabidopsis
and crop soybean
.