RNA-guided editing of bacterial genomes using CRISPR-Cas systems
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Zhang_RNA-guided editing.pdf
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Author(s)
Jiang, Wenyan
Bikard, David
Cox, David Daniel
Zhang, Feng
Marraffini, Luciano A.
Date Issued
January 2013
Journal
Nature Biotechnology
Publisher
Nature Publishing Group
Citation
Jiang, Wenyan, David Bikand, David Cox, Feng Zhang and Luciano A Marraffini. "RNA-guided editing of bacterial genomes using CRISPR-Cas systems." Nature Biotechnology 31 (2013) p.233–239.
Version
Author's final manuscript
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1038/nbt.2508
