Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation
Open accessAbstract
Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.
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The pattern of SpCas9-NG is similar to SpCas9-WT.
“The pattern of SpCas9-NG is similar to SpCas9-WT.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [43]
Our copy describes sgRNA sequence preferences and the NGG PAM required by S. pyogenes Cas9 (Abstract; page unavailable). Searches for SpCas9-NG, SpCas9-WT, and a comparison of engineered and wild-type Cas9 found no passage addressing whether their patterns are similar. (LLM, conf=0.76)
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- Authors
- Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B. L., Xavier, R. J., & Root, D. E.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Doench, John G
- Root, David E
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2014-09-03
- Volume
- 32
- Issue
- 12
- Pages
- 1262-1267
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4262738
- OpenAlex
- W1968342623
- Cited by
- 1,617
- References
- 25
- Citations by year
- 2026: 76 · 2025: 103 · 2024: 126 · 2023: 112 · 2022: 158 · 2021: 165 · 2020: 175 · 2019: 152 · 2018: 165 · 2017: 148 · 2016: 139 · 2015: 87 · 2014: 8 · 2013: 1
- FWCI
- 52.17
- 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)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (T32CA009172)
- National Institute of Allergy and Infectious Diseases (U19AI109725)
- National Institute of Diabetes and Digestive and Kidney Diseases (P30DK043351)
- 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 › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas9, single guide RNA design, gene inactivation, protospacer adjacent motif, SpCas9, null alleles, genetic screen
- MeSH terms
- Animals, Flow Cytometry, Humans, RNA, Streptococcus pyogenes, Gene Targeting, Gene Silencing, Mice, CRISPR-Cas Systems
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