Genome engineering using the CRISPR-Cas9 system
Open accessAbstract
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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Genome editing technology such as CRISPR promises to be the most important translational perspective in the future.
“Moreover, genome editing technology (e.g. the clustered regularly interspaced short palindromic repeats (CRISPR) technology) promises to be the most important translational perspective in the future [419–421].”
From Cardiovascular disease models: A game changing paradigm in drug discovery and screening (2019) · cites this paper as [421]
Plasmid-based CRISPR/Cas9 system is an appealing delivery approach owing to the simplicity and low cost of manipulation.
“The third option is the plasmid-based CRISPR/Cas9 system [93]. This is an appealing delivery approach owing to the simplicity and low cost of manipulation.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [93]
Different cell types or targeted genes can affect efficiency of CRISPR/Cas9 system.
“Meanwhile, different cell types or targeted genes can also affect the efficiency of the CRISPR/Cas9 system [93, 199].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [93]
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- Authors
- Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Zhang, Feng
- Venue
- Nature Protocols
- Published
- 2013-10-24
- Volume
- 8
- Issue
- 11
- Pages
- 2281-2308
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC3969860
- OpenAlex
- W1977709885
- Cited by
- 11,133
- References
- 76
- Citations by year
- 2026: 586 · 2025: 971 · 2024: 1018 · 2023: 1029 · 2022: 1148 · 2021: 1242 · 2020: 1232 · 2019: 1130 · 2018: 974 · 2017: 804 · 2016: 566 · 2015: 289 · 2014: 137 · 2013: 1
- FWCI
- 148.15
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Science Foundation (T32GM007753)
- Damon Runyon Cancer Research Foundation
- Fund for Astrophysical Research
- McKnight Foundation
- National Institutes of Health (R01‐grant)
- National Institute of Mental Health (DP1MH100706)
- National Institute of General Medical Sciences (T32GM008313, T32GM007753)
- National Institute of Diabetes and Digestive and Kidney Diseases (F32DK096822, R01DK097768, 1R01-DK097768)
- National Center for Chronic Disease Prevention and Health Promotion (1DP1-MH100706)
- 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, genome editing, guide RNA design, Cas9 nickase, off-target cleavage, non-homologous end joining, homology-directed repair, target site selection, cleavage efficiency, mammalian cells
- MeSH terms
- Base Sequence, Cell Line, Deoxyribonucleases, DNA Mutational Analysis, DNA Repair, Genetic Engineering, Humans, Molecular Sequence Data, Transfection, Mutagenesis, Genome, Cell Culture Techniques, HEK293 Cells, DNA End-Joining Repair, Genotyping Techniques, Clustered Regularly Interspaced Short Palindromic Repeats
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