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Bacterial genome engineering using CRISPR-associated transposases

Nature Protocols volume 19, pages 752–790 (2024)Cite this article

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in Escherichia coli at efficiencies approaching ~100%. Moreover, they generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1–2 weeks using available plasmid constructs and standard molecular biology techniques.

Key points

  • The protocol describes a novel and versatile CRISPR-associated transposase (CAST) system for the targeted and precise insertion of large DNA payloads into bacterial genomes.

  • Compared with pre-existing methods, this approach allows single and multiplexed insertion events at a desired location, with increased efficiency, reduced population heterogeneity, and improved specificity.

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Fig. 1: Overview of CASTs.
Fig. 2: Architecture of VchCAST vector constructs.
Fig. 3: Possible applications of CAST systems for microbial engineering.
Fig. 4: General CAST engineering workflow.
Fig. 5: PCR and qPCR analysis of integration products.
Fig. 6: Anticipated results from a CAST editing experiment.
Fig. 7: Alternative integration byproducts.
Fig. 8: Cloning CRISPR spacers for CAST systems.

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Data availability

Next-generation sequencing (NGS) data used for Figs. 6 and 7 are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (BioProject accession code PRJNA668381). Published genomes used for Tn-seq analyses in Fig. 6 were obtained from the NCBI (accessions codes CP001509.3).

Code availability

The CAST guide RNA design tool and associated documentation are available online via GitHub (https://github.com/sternberglab/CAST-guide-RNA-tool). Custom Python scripts used for the described Tn-seq NGS data analyses used in Fig. 6 are available online via GitHub (https://github.com/sternberglab/Vo_etal_2020).

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Acknowledgements

We thank S. Pesari for laboratory support, J. Mohabir and S. Acree for assistance with NGS read alignment, and the JP Sulzberger Columbia Genome Center for NGS support. H.H.W. acknowledges funding support from the National Science Foundation (MCB-2025515), National Institutes of Health (1R01EB031935, 2R01AI132403, 1R01DK118044 and 1R21AI146817), Burroughs Wellcome Fund (1016691) and Department of Defense Army Research Office (W911NF-22-2-0210). D.R.G. is supported by the Burroughs Wellcome Fund Postdoctoral Diversity Enrichment Program. S.H.S. acknowledges funding support from the National Institutes of Health (DP2HG011650, R21AI168976, R01EB031935, and R01EB027793), the Pew Biomedical Scholars Program, the Alfred Sloan Foundation Research Fellowship, the Irma T. Hirschl Career Scientist Award, and a generous startup package from the Columbia University Irving Medical Center Dean’s Office and the Vagelos Precision Medicine Fund.

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Author notes

  1. Phuc Leo H. Vo

    Present address: Vertex Pharmaceuticals, Inc, Boston, MA, USA

  2. Sanne E. Klompe

    Present address: Department of Genomes and Genetics, Institut Pasteur, Paris, France

  3. Carlotta Ronda

    Present address: Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Diego Rivera Gelsinger, Sanne E. Klompe & Samuel H. Sternberg

  2. Department of Systems Biology, Columbia University, New York, NY, USA

    Diego Rivera Gelsinger, Carlotta Ronda & Harris H. Wang

  3. Department of Molecular Pharmacology and Therapeutics, Columbia University, New York, NY, USA

    Phuc Leo H. Vo

  4. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Harris H. Wang

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Contributions

D.R.G., P.L.H.V. and S.H.S. wrote the manuscript. D.R.G., P.L.H.V. and S.E.K. designed the figures, and C.R. and H.H.W. discussed figure ideas, provided information on conjugations, and provided feedback on the manuscript.

Corresponding author

Correspondence to Samuel H. Sternberg.

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Competing interests

Columbia University has filed patent applications related to this work. S.H.S. is a cofounder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits. H.H.W. is a scientific advisor of SNIPR Biome, Kingdom Supercultures, Fitbiomics, Arranta Bio, VecX Biomedicines and Genus PLC, and a scientific cofounder of Aclid, none of whom are involved in the study. The remaining authors declare no competing interests.

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Key references using this protocol

Vo, P. L. H. et al. Nat. Biotechnol. 39, 480–489 (2021): https://doi.org/10.1038/s41587-020-00745-y

Hoffmann, F. T. et al. Nature 609, 384–393 (2022): https://doi.org/10.1038/s41586-022-05059-4

Walker, M. W. G. et al. Nucleic Acids Res. 51, 4519–4535 (2023): https://doi.org/10.1093/nar/gkad270

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Gelsinger, D.R., Vo, P.L.H., Klompe, S.E. et al. Bacterial genome engineering using CRISPR-associated transposases. Nat Protoc 19, 752–790 (2024). https://doi.org/10.1038/s41596-023-00927-3

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