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Cited by 2,098CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

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RNA-programmed genome editing in human cells

Open access
Jinek, Martin, East, Alexandra, Cheng, Aaron, Lin, Steven, Ma, Enbo, Doudna, Jennifer·2013·eLife
DOI 10.7554/elife.00471PMC PMC3557905
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Abstract

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.

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Figure 1. Co-expression of Cas9 and guide RNA in human cells generates double-strand DNA breaks at the target locus. (A) Top: schematic diagram of the Cas9-HA-NLS-GFP expression construct. Bottom: lysate from HEK293T cells transfected with the Cas9 expression plasmid was analyzed by Western blotting using an anti-HA antibody. (B) Fluorescence microscopy of HEK293T cells expressing Cas9-HA-NLS-GFP. (C) Blue line indicates the sequence used for the guide segment of CLTA1 sgRNA. Top: schematic diagram of the sgRNA target site in exon 7 of the human CLTA gene. The target sequence that hybridizes to the guide segment of CLTA1 sgRNA is indicated by the blue line. The GG nucleotide protospacer adjacent motif (PAM) is highlighted in yellow. Black lines denote the DNA binding regions of the control ZFN protein. The translation stop codon of the CLTA open reading frame is highlighted in red for reference. Middle: schematic diagram of the sgRNA expression construct. The RNA is expressed under the control of the U6 Pol III promoter and a poly(T) tract that serves as a Pol III transcriptional terminator signal. Bottom: sgRNA-guided cleavage of target DNA by Cas9. The sgRNA consists of a 20-nt 5′-terminal guide segment (blue) followed by a 42-nt stem-loop structure required for Cas9 binding (red). Cas9-mediated cleavage of the two target DNA strands occurs upon unwinding of the target DNA and formation of a duplex between the guide segment of the sgRNA and the target DNA. This is dependent on the presence of a GG dinucleotide PAM downstream of the target sequence in the target DNA. Note that the target sequence is inverted relative to the upper diagram. (D) Northern blot analysis of sgRNA expression in HEK239T cells. (E) Surveyor nuclease assay of genomic DNA isolated from HEK293T cells expressing Cas9 and/or CLTA sgRNA. A ZFN construct previously used to target the CLTA locus (Doyon et al., 2011) was used as a positive control for detecting DSB-induced DNA repair by non-homologous end joining. DOI: http://dx.doi.org/10.7554/eLife.00471.003
Figure 1. Co-expression of Cas9 and guide RNA in human cells generates double-strand DNA breaks at the target locus. (A) Top: schematic diagram of the Cas9-HA-NLS-GFP expression construct. Bottom: lysate from HEK293T cells transfected with the Cas9 expression plasmid was analyzed by Western blotting using an anti-HA antibody. (B) Fluorescence microscopy of HEK293T cells expressing Cas9-HA-NLS-GFP. (C) Blue line indicates the sequence used for the guide segment of CLTA1 sgRNA. Top: schematic diagram of the sgRNA target site in exon 7 of the human CLTA gene. The target sequence that hybridizes to the guide segment of CLTA1 sgRNA is indicated by the blue line. The GG nucleotide protospacer adjacent motif (PAM) is highlighted in yellow. Black lines denote the DNA binding regions of the control ZFN protein. The translation stop codon of the CLTA open reading frame is highlighted in red for reference. Middle: schematic diagram of the sgRNA expression construct. The RNA is expressed under the control of the U6 Pol III promoter and a poly(T) tract that serves as a Pol III transcriptional terminator signal. Bottom: sgRNA-guided cleavage of target DNA by Cas9. The sgRNA consists of a 20-nt 5′-terminal guide segment (blue) followed by a 42-nt stem-loop structure required for Cas9 binding (red). Cas9-mediated cleavage of the two target DNA strands occurs upon unwinding of the target DNA and formation of a duplex between the guide segment of the sgRNA and the target DNA. This is dependent on the presence of a GG dinucleotide PAM downstream of the target sequence in the target DNA. Note that the target sequence is inverted relative to the upper diagram. (D) Northern blot analysis of sgRNA expression in HEK239T cells. (E) Surveyor nuclease assay of genomic DNA isolated from HEK293T cells expressing Cas9 and/or CLTA sgRNA. A ZFN construct previously used to target the CLTA locus (Doyon et al., 2011) was used as a positive control for detecting DSB-induced DNA repair by non-homologous end joining. DOI: http://dx.doi.org/10.7554/eLife.00471.003CC BY · PubMed Central
Figure 1—figure supplement 1. Mutated alleles of the CLTA gene in HEK293T cells as a result of Cas9-induced NHEJ. Four types of indels were identified by Sanger sequencing in 68 clonal amplicons. The protospacer sequence is highlighted in grey with the putative cleavage site indicated by the arrow. Dashes represent deleted bases. DOI: http://dx.doi.org/10.7554/eLife.00471.004
Figure 1—figure supplement 1. Mutated alleles of the CLTA gene in HEK293T cells as a result of Cas9-induced NHEJ. Four types of indels were identified by Sanger sequencing in 68 clonal amplicons. The protospacer sequence is highlighted in grey with the putative cleavage site indicated by the arrow. Dashes represent deleted bases. DOI: http://dx.doi.org/10.7554/eLife.00471.004CC BY · PubMed Central
Figure 2. Cell lysates contain active Cas9:sgRNA and support site-specific DNA cleavage. (A) Lysates from cells transfected with the plasmid(s) indicated at left were incubated with plasmid DNA containing a PAM and the target sequence complementary to the CLTA1 sgRNA; where indicated, the reaction was supplemented with 10 pmol of in vitro transcribed CLTA1 sgRNA; secondary cleavage with XhoI generated fragments of ∼2230 and ∼3100 bp fragments indicative of Cas9-mediated cleavage. A control reaction using lysate from cells transfected with a ZFN expression construct shows fragments of slightly different size reflecting the offset of the ZFN target site relative to the CLTA1 target site. (B) Lysates from cells transfected with Cas9-HA-NLS-GFP expression plasmid and, where indicated, the CLTA1 sgRNA expression plasmid, were incubated with target plasmid DNA as in (A) in the absence or presence of in vitro-transcribed CLTA1 sgRNA. DOI: http://dx.doi.org/10.7554/eLife.00471.005
Figure 2. Cell lysates contain active Cas9:sgRNA and support site-specific DNA cleavage. (A) Lysates from cells transfected with the plasmid(s) indicated at left were incubated with plasmid DNA containing a PAM and the target sequence complementary to the CLTA1 sgRNA; where indicated, the reaction was supplemented with 10 pmol of in vitro transcribed CLTA1 sgRNA; secondary cleavage with XhoI generated fragments of ∼2230 and ∼3100 bp fragments indicative of Cas9-mediated cleavage. A control reaction using lysate from cells transfected with a ZFN expression construct shows fragments of slightly different size reflecting the offset of the ZFN target site relative to the CLTA1 target site. (B) Lysates from cells transfected with Cas9-HA-NLS-GFP expression plasmid and, where indicated, the CLTA1 sgRNA expression plasmid, were incubated with target plasmid DNA as in (A) in the absence or presence of in vitro-transcribed CLTA1 sgRNA. DOI: http://dx.doi.org/10.7554/eLife.00471.005CC BY · PubMed Central
Figure 3. 3′ extension of sgRNA constructs enhances site-specific NHEJ-mediated mutagenesis. (A) The construct for CLTA1 sgRNA expression (top) was designed to generate transcripts containing the original Cas9-binding sequence v1.0 (Jinek et al., 2012), or sequences extended by 4 base pairs (v2.1) or 10 base pairs (v2.2). (B) Surveyor nuclease assay of genomic DNA isolated from HEK293T cells expressing Cas9 and/or CLTA sgRNA v1.0, v2.1 or v2.2. A ZFN construct previously used to target the CLTA locus (Doyon et al., 2011) was used as a positive control for detecting DSB-induced DNA repair by non-homologous end joining. DOI: http://dx.doi.org/10.7554/eLife.00471.006
Figure 3. 3′ extension of sgRNA constructs enhances site-specific NHEJ-mediated mutagenesis. (A) The construct for CLTA1 sgRNA expression (top) was designed to generate transcripts containing the original Cas9-binding sequence v1.0 (Jinek et al., 2012), or sequences extended by 4 base pairs (v2.1) or 10 base pairs (v2.2). (B) Surveyor nuclease assay of genomic DNA isolated from HEK293T cells expressing Cas9 and/or CLTA sgRNA v1.0, v2.1 or v2.2. A ZFN construct previously used to target the CLTA locus (Doyon et al., 2011) was used as a positive control for detecting DSB-induced DNA repair by non-homologous end joining. DOI: http://dx.doi.org/10.7554/eLife.00471.006CC BY · PubMed Central

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Metadata

Authors
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J.
Affiliations
  • Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
  • University of California, Berkeley (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
Doudna, Jennifer
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2013-01-29
Volume
2
Pages
e00471-e00471
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.00471
PMC
PMC3557905
OpenAlex
W2153344788
Cited by
2,098
References
14
Citations by year
2026: 36 · 2025: 83 · 2024: 104 · 2023: 123 · 2022: 174 · 2021: 159 · 2020: 191 · 2019: 181 · 2018: 196 · 2017: 203 · 2016: 226 · 2015: 174 · 2014: 153 · 2013: 95
FWCI
96.82
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, other-oa)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion, other-oa)
  • Zurich Open Repository and Archive (University of Zurich)
Funders
  • Howard Hughes Medical Institute
  • National Institutes of Health
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, Cas9, DNA double-strand breaks, site-specific DNA cleavage, DNA targeting, human cells, genome editing
MeSH terms
Humans, RNA, RNA Editing, Clustered Regularly Interspaced Short Palindromic Repeats

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