CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5′ nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.
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We thank L. T. Hille, C. R. R. Alves, and R. A. Silverstein for suggestions about the manuscript, P. M. Boone for assistance with ddPCR, and B. L. Stoddard for advice. C.J.T. was supported by a National Science Foundation Graduate Research Fellowship grant number 2020295403. B.P.K. was supported by a Mass General Hospital Howard M. Goodman Fellowship.
C.J.T. and B.P.K are inventors on patents and/or patent applications filed by Massachusetts General Brigham that describe genome-engineering technologies. B.P.K. is a consultant for EcoR1 capital, and is an advisor to Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine. B.O. declares no competing interests.
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Tou, C.J., Orr, B. & Kleinstiver, B.P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-022-01574-x