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Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases

Nature Biotechnology volume 41, pages 968–979 (2023)Cite this article

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Abstract

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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Fig. 1: Development and characterization of HELIX.
Fig. 2: Characterization of DNA insertions on genomic targets using HELIX.
Fig. 3: Extension of HELIX to type V-K CAST orthologs.
Fig. 4: Specificity profiling of ShCAST and ShHELIX systems.
Fig. 5: HELIX-mediated DNA insertion in human cell lysates and human cells.

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Data availability

Sequencing data has been deposited with the NCBI Sequence Read Archive (SRA) under BioProject ID PRJNA889059. All other primary datasets are available in Supplementary Table 4.

Code availability

A custom script was used to calculate on-target integration from specificity analyses and is available in Supplementary Note 6. All other analyses were performed using publicly avaliable software noted in the Methods.

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Acknowledgements

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.

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Authors and Affiliations

  1. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Connor J. Tou, Benno Orr & Benjamin P. Kleinstiver

  2. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Connor J. Tou, Benno Orr & Benjamin P. Kleinstiver

  3. Biological Engineering Program, Massachusetts Institute of Technology, Cambridge, MA, USA

    Connor J. Tou

  4. Biological and Biomedical Sciences Program, Harvard University, Boston, MA, USA

    Benno Orr

  5. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Benjamin P. Kleinstiver

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Contributions

C.J.T conceived of the idea, designed and performed experiments, and wrote the manuscript draft. B.O. conducted experiments for ShoHELIX and cargo size comparisons. B.P.K supervised the study and contributed to experimental design. C.J.T and B.P.K wrote and revised the manuscript with input from B.O.

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Correspondence to Benjamin P. Kleinstiver.

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Competing interests

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 41, 968–979 (2023). https://doi.org/10.1038/s41587-022-01574-x

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