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Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration


Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in E. coli requires CRISPR- and transposon-associated molecular machineries, including a novel co-complex between Cascade and the transposition protein TniQ. Donor DNA integration occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep sequencing experiments reveal highly specific, genome-wide DNA integration across dozens of unique target sites. This work provides the first example of a fully programmable, RNA-guided integrase and lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.

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Author information

Correspondence to Samuel H. Sternberg.

Supplementary information

Supplementary Note

Nomenclature for transposons and CRISPR-Cas systems described in this study.

Reporting Summary

Supplementary Figures

This file contains Supplementary Figures 1-8 including legends.

Supplementary Table 1

Description and sequence of plasmids used in this study.

Supplementary Table 2

Gene and protein sequences for the Vibrio cholerae RNA-guided DNA integration machinery used in this study. * The V. cholerae HE-45 genome contains another Tn7-like transposon (GenBank accession ALED01000025.1), which lacks an encoded CRISPR–Cas system and exhibits low sequence similarity to the transposon investigated in this study. † The gene sequences shown are copied from the Vibrio cholerae HE-45 genome. Actual sequences used in this study contained additional silent point mutations for cloning purposes, and can be found in Supplementary Table 1. ‡ The protein sequences shown are full-length translations from the Vibrio cholerae HE-45 genome. TnsA in our experiments contained an additional alanine residue after the N-terminal methionine. § Cas8 is a Cas8-Cas5 fusion protein, as described in the main text.

Supplementary Table 3

Guide RNAs and genomic target sites used in this study. * Coordinates are for the E. coli BL21(DE3) genome (GenBank accession CP001509). † PAM sequences denote the 2 nucleotides immediately 5’ of the target (V. cholerae and P. aeruginosa Cascade) or 3 nucleotides immediately 3’ of the target (S. pyogenes Cas9) on the non-target strand.

Supplementary Table 4

Next-generation sequencing library statistics.

Supplementary Table 5

Oligonucleotides used for PCR, qPCR, and NGS experiments in this study.

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