Nat Biotechnol

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.1DP2AI104556-01/AI/NIAID NIH HHS/United StatesDP1 MH100706/MH/NIMH NIH HHS/United StatesDP1MH100706/DP/NCCDPHP CDC HHS/United StatesDP2 AI104556/AI/NIAID NIH HHS/United StatesR01/PHS HHS/United StatesT32 GM007753/GM/NIGMS NIH HHS/United States2013-09-01T00:00:00Z23360965PMC374894