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Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration


The adaptation phase of CRISPR–Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1–Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1–Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

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Fig. 1: Whole-genome deep sequencing reveals off-target spacer integration events within the E. coli genome.
Fig. 2: Spacer-seq identifies hundreds of off-target spacer integration sites within the E. coli genome.
Fig. 3: Effects of genomic knockouts of IHF and the CRISPR1 locus on off-target spacer integration activity.
Fig. 4: Comparison of three different NCA sequences and their activity in target interference and primed acquisition.
Fig. 5: Evidence for native off-target spacer integrations.

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The authors thank Ekaterina Semonova and Konstantin Severinov (Rutgers) for generously providing strain BW40114, and J. Doudna and L. Harrington (UCB) for generously providing BL21-AI IHF-knockout strains. The project was supported by grants from the National Human Genome Research Institute (5R01MH103910), the National Human Genome Research Institute (5RM1HG008525) and the Simons Foundation Autism Research Initiative (368485) to G.M.C.

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J.N. and S.L.S. conceived the study. J.N. designed the work, performed the experiments, analysed the data and wrote the manuscript with input from S.L.S. and G.M.C. S.L.S. and G.M.C. discussed the results and commented on the manuscript.

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Correspondence to George M. Church.

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J.N., S.L.S. and G.M.C. are inventors on a provisional patent (62/490,901) filed by the President and Fellows of Harvard College that covers the work in this manuscript. A complete account of the financial interests of G.M.C. is listed at:

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Nivala, J., Shipman, S.L. & Church, G.M. Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration. Nat Microbiol 3, 310–318 (2018).

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