Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
Open accessAbstract
The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.
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Cell-penetrating peptide-mediated delivery of CRISPR-Cas9 into cells increases editing efficiency, but the optimal editing efficiency remains under 20% in vitro.
“Though cell-penetrating peptidemediated delivery of CRISPR-Cas9 into cells was reported to increase editing efficiency, the optimal editing efficiency was still under 20% in vitro.59”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [59]
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- Authors
- Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J., & Church, G. M.
- Affiliations
- Harvard University (US)
- Corresponding author
- Church, George M
- Venue
- Nature Methods
- Published
- 2013-09-29
- Volume
- 10
- Issue
- 11
- Pages
- 1116-1121
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC3844869
- OpenAlex
- W4206892629
- Cited by
- 786
- References
- 31
- Citations by year
- 2026: 11 · 2025: 31 · 2024: 34 · 2023: 36 · 2022: 46 · 2021: 56 · 2020: 67 · 2019: 71 · 2018: 70 · 2017: 101 · 2016: 98 · 2015: 84 · 2014: 74 · 2013: 5
- FWCI
- 38.94
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Funders
- U.S. Department of Energy (DE‐FG02‐, DEFG0202ER63445, FG02-02ER63445)
- National Institutes of Health (DE-FG02-02ER63445)
- Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
- National Human Genome Research Institute (P50HG005550)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- Cas9 endonuclease, Streptococcus pyogenes, Neisseria meningitidis, CRISPR/Cas9, RNA-guided genome editing, human cells, bacteria
- MeSH terms
- Amino Acid Sequence, Bacterial Proteins, Gene Expression Regulation, Molecular Sequence Data, RNA, Streptomyces, Sequence Homology, Amino Acid, RNA Editing
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