CRISPR-assisted editing of bacterial genomes

Details

• Alternative Title:
  Nat Biotechnol
• Personal Author:
  Jiang, Wenyan ; Bikard, David ; Cox, David ; Zhang, Feng ; Marraffini, Luciano A.
• Description:
  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.
• Subjects:
  Base Sequence DNA Cleavage Endonucleases Escherichia Coli Genetic Engineering Genome, Bacterial Molecular Sequence Data Mutagenesis, Site-Directed RNA, Small Untranslated Streptococcus Pneumoniae
• Source:
  Nat Biotechnol. 31(3):233-239
• Pubmed ID:
  23360965
• Pubmed Central ID:
  PMC3748948
• Document Type:
  Journal Article
• Funding:
  1DP2AI104556-01/AI/NIAID NIH HHSUnited States/ ; T32 GM007753/GM/NIGMS NIH HHSUnited States/ ; DP1 MH100706/MH/NIMH NIH HHSUnited States/ ; DP2 AI104556/AI/NIAID NIH HHSUnited States/ ; R01/PHS HHSUnited States/ ; DP1MH100706/DP/NCCDPHP CDC HHSUnited States/
• Volume:
  31
• Issue:
  3
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:e5ac44f538771fa51acf47d86a1642e9dd70291cebe3e2f97b8dec523722ed23
• Download URL:
  https://stacks.cdc.gov/view/cdc/22571/cdc_22571_DS1.pdf
• File Type:
  [PDF - 1.78 MB ]