Multiplex Genome Engineering Using CRISPR/Cas Systems
Open accessAbstract
Genome Editing: Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost ). Cong et al. (p. 819 , published online 3 January) and Mali et al. (p. 823 , published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells.
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CRISPR/Cas9 is a class of ribonucleoprotein complexes formed by a Cas9 protein and a single guide RNA (sgRNA).
“CRISPR/Cas9 is a class of ribonucleoprotein complexes formed by a Cas9 protein and a single guide RNA (sgRNA) (Jinek et al., 2012; Cho et al., 2013; Cong et al., 2013; Wang et al., 2016a).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cong et al., 2013
The paper describes Cas9 guided by a chimeric RNA that fuses crRNA and tracrRNA, supporting the single-guide-RNA version of the system (section labeled Abstract; page unavailable). But it describes the natural system as using two RNAs and identifies Cas9 plus those two RNAs as the minimal system tested, so it does not support characterizing CRISPR/Cas9 generally as consisting of Cas9 and one sgRNA. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe paper notes that adapting bacterial CRISPR systems for RNA-guided cleavage of complementary DNA transformed mammalian gene editing.
“Repurposing bacterial “CRISPR” systems to facilitate RNA-guided cleavage of complementary DNA has revolutionized the field of gene editing in mammalian cells 1–4.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [2]
Target specificity of the CRISPR/Cas system is very high and a single mutation can disrupt targeting.
“Target specificity of the CRISPR/Cas system is very high and a single mutation can disrupt targeting 20”
From Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus (2013) · cites this paper as [20]
Cas9 can create DNA cleavage at desired genomic positions guided by precise base pairing between sgRNA and DNA, adjacent to a protospacer-adjacent motif (PAM).
“Cas9 can create DNA cleavage at desired genomic positions that are guided by precise base pairing between sgRNA and DNA, adjacent to a protospacer-adjacent motif (PAM) (Jinek et al., 2012; Cho et al., 2013; Cong et al., 2013; Wang et al., 2016a).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cong et al., 2013
Mammalian genomes can be edited by homologous recombination when a double-strand break is introduced and a DNA template with high sequence similarity is provided; an artificial template can incorporate desired DNA sequences.
“When a double-stranded break is introduced into genomic DNA, cells can repair the break by using a DNA molecule with high sequence similarity as a template. If an artificial template is provided while the DNA break is introduced, this pathway provides a mean to incorporate desired DNA sequences into the genome34,35”
From CRISPR-based functional genomics for neurological disease (2020) · cites this paper as [34]
A simple two-component system of Streptococcus pyogenes Cas9 bound to a single guide RNA can function as a programmable nuclease that can cleave specific sequences in mammalian genomes.
“The discovery, made less than 10 years ago, that a simple two-component system of a Cas protein (Streptococcus pyogenes Cas9) bound to a single guide RNA (sgRNA) can function as a programmable nuclease33 that can be deployed to cleave specific sequences in mammalian genomes34,35 has been transformative for biomedical research and the development of gene therapies.”
From CRISPR-based functional genomics for neurological disease (2020) · cites this paper as [34]
The CRISPR/Cas9 system has been used for multiplex genome cleavage.
“The CRISPR/Cas9 system has been used for multiplex genome cleavage.20”
From The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo (2014) · cites this paper as [20]
The CRISPR/Cas system has been successfully applied to human cells and several model organisms for genome editing.
“Recently, the CRISPR/Cas system has been successfully applied to human cells19,20,21,22 and several model organisms for genome editing.23,24,25,26”
From The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo (2014) · cites this paper as [20]
LTR-A- and -B-directed 30-bp protospacer and pre-crRNA system were selected to enhance specificity/efficiency versus 20-bp protospacer-based chimeric crRNA-tracRNA system.
“selection of LTR-A-and -B-directed, 30-bp protospacer and also precrRNA system reflecting the original bacterial immune mechanism to enhance specificity/efficiency vs. 20-bp protospacer-based, chimeric crRNA-tracRNA system”
From RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection (2014) · cites this paper as [16]
Cas9 tolerates mismatches between the guide RNA and target DNA in a sequence-dependent manner and potentially tolerates up to five target mismatches in human cells, thereby promoting undesired off-target mutations.
“as mentioned in several recent publications, Cas9 tolerates mismatches between the guide RNA and target DNA in a sequence-dependent manner and potentially tolerates up to five target mismatches in human cells [12], [24], [25], [26], [27], thereby promoting undesired off-target mutations.”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [12]
The sequence specificity of Streptococcus pyogenes Cas9 tolerates 4 mismatches between the gRNA and complementary sequence.
“Because the sequence specificity of Streptococcus pyogenes Cas9 tolerates 4 mismatches between the gRNA and complementary sequence”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [12]
Compared with ZFNs and TALENs, the new CRISPR-Cas approach is more convenient and efficient.
“Compared with well-known, site-specific genome editing technologies, such as ZFNs and TALENs, the new CRISPR-Cas approach is more convenient and efficient [10], [12].”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [12]
Non-specific cleavage may occur in the cellular genome.
“non-specific cleavage may occur in the cellular genome [12], [24], [25], [26], [27].”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [12]
CRISPR/Cas9 is a naturally occurring bacterial immune system that protects bacteria from phage infection and has been harnessed for genome-engineering applications.
“Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a naturally occurring bacterial immune system that uses a novel nuclease system to protect bacteria from phage infection (6-9), and it has recently been harnessed for a variety of genome-engineering applications (10)(11)(12)(13)(14)(15)(16)(17).”
From RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection (2014) · cites this paper as [11]
In available publications using CRISPR/Cas9 system, the gene editing frequency is comparable with ZFN or TALEN.
“In the few available publications using CRISPR/Cas9 system, the gene editing frequency is comparable with ZFN or TALEN [87].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [87]
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 [87]
CRISPR/Cas9 system can be easily employed to target multiple sites to induce a deletion of a large gene fragment.
“As described by Zhang group [87], CRISPR/Cas 9 system can be easily employed to target multiple sites to induce a deletion of a large gene fragment.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [87]
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 [87]
No significant cytotoxicity was observed with CRISPR/Cas9.
“Moreover, no significant cytotoxicity was observed [87,88].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [87]
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“While our paper was under review, several very recent studies reported the use of wild-type Cas9/CRISPR for targeted genome editing in diverse organisms (Mali et al., 2013; Cong et al., 2013; Jinek et al., 2013; Jiang et al., 2013; Hwang et al., 2013; Cho et al., 2013).”
BackgroundSearch-and-replace genome editing without double-strand breaks or donor DNA
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- Authors
- Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Columbia University (US)
- Koch Institute for Integrative Cancer Research At MIT (US)
- Rockefeller University (US)
- Corresponding author
- Cong, Le
- Ran, F. Ann
- Zhang, Feng
- ORCID
- Venue
- Science
- Published
- 2013-02-15
- Volume
- 339
- Issue
- 6121
- Pages
- 819-823
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC3795411
- OpenAlex
- W2064815984
- Semantic Scholar
- 51bcf2a1577b3dd876fcaa4cdf630f54d261c107
- Cited by
- 15,339
- References
- 32
- Citations by year
- 2026: 614 · 2025: 926 · 2024: 1041 · 2023: 1127 · 2022: 1311 · 2021: 1421 · 2020: 1456 · 2019: 1399 · 2018: 1464 · 2017: 1368 · 2016: 1261 · 2015: 888 · 2014: 732 · 2013: 305 · 2012: 5
- FWCI
- 476.05
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- PMC
- Funders
- Howard Hughes Medical Institute
- National Institute of Mental Health (DP1MH100706)
- National Cancer Institute (R01CA133404)
- National Institute of General Medical Sciences (R01GM034277, R01 GM34277, T32GM007753)
- National Institute of Allergy and Infectious Diseases (DP2AI104556)
- National Institute of Neurological Disorders and Stroke (R01NS073124)
- 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 › Molecular Biology › RNA regulation and disease
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, genome editing, RNA-guided DNA cleavage, nicking enzyme, homology-directed repair, single guide RNA, CRISPR arrays, human cells, mouse cells, mutagenesis
- MeSH terms
- Animals, Base Sequence, DNA, Genetic Engineering, Humans, Molecular Sequence Data, RNA, Streptococcus pyogenes, Mutagenesis, Genome, Microarray Analysis, Mice, DNA Cleavage, Inverted Repeat Sequences, Genetic Loci, Recombinational DNA Repair, CRISPR-Cas Systems
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