Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
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We thank V. Fischetti and C. Euler for plasmid pLZ12spec, D. Court for the HME63 strain, J. Kern for plasmid pKD46, the Rockefeller University Genomic Resource Center for technical support and J. Roberts for the MG1655 strain. D.B. is supported by a Harvey L. Karp Discovery Award and the Bettencourt Schuller Foundation. D.C. is supported by the Medical Scientist Training Program. F.Z. is supported by a US National Institutes of Health (NIH) Director's Pioneer Award (DP1MH100706), Transformative R01, the Keck, McKnight, Gates, Damon Runyon, Searle Scholars, Klingenstein, and Simons Foundations, Bob Metcalfe, Mike Boylan and Jane Pauley. L.A.M. is supported by the Searle Scholars Program, the Rita Allen Scholars Program, a Irma T. Hirschl Award and a NIH Director's New Innovator Award (1DP2AI104556-01).
The authors declare no competing financial interests.
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Jiang, W., Bikard, D., Cox, D. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31, 233–239 (2013). https://doi.org/10.1038/nbt.2508
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