The DOI matches its Crossref record; 76 of the 76 references it cites were found in a registry or an index.
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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“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]
In the citing paper
“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]
In the citing paper
“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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Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339
Self-citationRNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210
CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A., & Horvath, P. (2007) · Science · doi:10.1126/science.1138140 · Cited by 5,947
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
Self-citationCRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., et al. (2013) · Cell · doi:10.1016/j.cell.2013.06.044 · Cited by 3,696
Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013) · Cell · doi:10.1016/j.cell.2013.04.025 · Cited by 3,495
Self-citationDouble nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013) · Cell · doi:10.1016/j.cell.2013.08.021 · Cited by 3,299
Self-citationHigh-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292
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Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016) · Nature · doi:10.1038/nature17946 · Cited by 5,437
Gene regulation by long non-coding RNAs and its biological functions
Statello, L., Guo, C.-J., Chen, L.-L., & Huarte, M. (2021) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-020-00315-9 · Cited by 4,991
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153
Easy quantitative assessment of genome editing by sequence trace decomposition
Brinkman, E. K., Chen, T., Amendola, M., & van Steensel, B. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku936 · Cited by 2,390
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268
Metadata
- 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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