Bacterial genome engineering using CRISPR-associated transposases

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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.
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Funding Information
  • U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (EB031935, 1R01EB031935)
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (AI168976, 2R01AI132403)
  • U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (HG011650)
  • Pew Charitable Trusts (00034095)
  • Alfred P. Sloan Foundation (FG-2020-12881)
  • Irma T. Hirschl Trust (Career Scientist Award)
  • Burroughs Wellcome Fund (Postdoctoral Diversity Enrichment Program, 1016691)
  • U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering
  • U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (1R21AI146817, 1R01DK118044)
  • National Science Foundation (MCB-2025515)
  • U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases
  • U.S. Department of War (W911NF-22-2-0210)

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