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bioRxiv · 10.64898/2026.07.15.738807

CRISPR-Associated Transposases Enable Programmable DNA Integration in Plants

Wang, Y.·Muchenje, K.·Papikian, A.·Legendre, M.·Leem, E.·Demirer, G. S.
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Abstract

Programmable DNA integration is a major challenge in plant genome engineering. CRISPR-associated transposases (CAST) catalyze efficient RNA-guided DNA integration without double-strand breaks, yet their activity has not been established in plants. Here, we reconstituted and engineered a Type I-F CAST for programmable DNA integration in plant cells. We validated expression of the wild-type Pseudoalteromonas CAST (PseCAST) machinery in plants and established targeted episomal integration in Arabidopsis thaliana protoplasts and chromosomal integration at a transgenic locus in Nicotiana benthamiana. The evolved PseCAST system, evoCAST, showed chromosomal integration efficiencies of 2.7%, representing a 6-fold improvement over wild-type PseCAST. evoCAST also enabled the insertion of cis-regulatory elements into a synthetic landing pad with 8% efficiency. evoCAST was subsequently retargeted to six endogenous genomic loci, demonstrating programmable integration across diverse chromosomal contexts. Finally, a cofactor screen identified the chromatin-associated factor AtHMGB2 as an enhancer of evoCAST-mediated integration activity in plants. These results establish CAST as a functional platform for programmable DNA insertion in plants and provide a foundation for developing targeted genome-engineering technologies for crop biotechnology.

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Wang, Y., Muchenje, K., Papikian, A., Legendre, M., Leem, E., Demirer, G. S.. 2026-07-16. CRISPR-Associated Transposases Enable Programmable DNA Integration in Plants. https://doi.org/10.64898/2026.07.15.738807

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