RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Open accessAbstract
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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The type II CRISPR/Cas9 system has been exploited for high-efficiency gene editing in model microorganisms such as E. coli, S. cerevisiae, and Streptococcus pneumoniae.
“The type II CRISPR/Cas9 system has been exploited for high-efficiency gene editing in model microorganisms such as E. coli (Jiang et al., 2015), S. cerevisiae (DiCarlo et al., 2013), and Streptococcus pneumoniae (Jiang et al., 2013).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Jiang et al., 2013
Each base within the 20 nt guide sequence contributes to overall specificity.
“Collectively, these studies indicate that although each base within the 20 nt guide sequence contributes to overall specificity”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [93]
Multiple mismatches between the guide RNA and its complementary target DNA sequence can be tolerated depending on the quantity, position, and base identity of mismatches, contributing to potential off-target activity.
“multiple mismatches between the guide RNA and its complementary target DNA sequence can be tolerated depending on the quantity, position, and base identity of mismatches [87,90,91,93], all of which contribute to potential off-target activity.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [93]
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- Authors
- Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A.
- Affiliations
- Rockefeller University (US)
- Broad Institute (US)
- Corresponding author
- Bikard, David
- Marraffini, Luciano A
- Venue
- Nature Biotechnology
- Published
- 2013-01-29
- Volume
- 31
- Issue
- 3
- Pages
- 233-239
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3748948
- OpenAlex
- W2000046950
- Cited by
- 2,429
- References
- 51
- Citations by year
- 2026: 84 · 2025: 124 · 2024: 151 · 2023: 162 · 2022: 199 · 2021: 203 · 2020: 245 · 2019: 185 · 2018: 206 · 2017: 208 · 2016: 203 · 2015: 174 · 2014: 183 · 2013: 98
- FWCI
- 98.32
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- PMC
- Funders
- U.S. Public Health Service
- National Institute of Mental Health (DP1MH100706)
- National Institute of General Medical Sciences (T32GM007753)
- National Institute of Allergy and Infectious Diseases (DP2AI104556)
- National Center for Chronic Disease Prevention and Health Promotion (DP1‑MH100706)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas9, bacterial genome editing, Streptococcus pneumoniae, Escherichia coli, targeted mutagenesis, recombineering, genome editing
- MeSH terms
- Base Sequence, Endonucleases, Escherichia coli, Genetic Engineering, Molecular Sequence Data, Streptococcus pneumoniae, Mutagenesis, Site-Directed, Genome, Bacterial, DNA Cleavage, RNA, Small Untranslated
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