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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., (© 2024. Springer Nature Limited.)

Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering., Competing Interests: Declaration of interests Columbia University has filed a patent application related to this work for which S.E.K. and S.H.S. are inventors. S.E.K. and S.H.S. are inventors on other patents and patent applications related to CRISPR-Cas systems and uses thereof. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to Prime Medicine and CrisprBits, and an equity holder in Dahlia Biosciences and Caribou Biosciences., (Copyright © 2021 Elsevier Inc. All rights reserved.)