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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
Open accessAbstract
Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
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“The markedly high efficiency of CRISPR/Cas systems coupled with the above-mentioned advantages over other methods justify the CRISPR “epidemic” that the scientific community experienced in 2013 [62,81,100,101].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [101]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“Scientists engineered a double nicking scheme featuring a pair of Cas9n targeting opposite strands where neighbouring gRNA targets are offset by a certain number of base pairs [101].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [101]
In the citing paper
“Consistent with a previous report 19 , this suggests that TALENs can tolerate two mismatches in vivo.”
From TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis (2014) · cites this paper as [19]
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“However, it has been recently shown that CRISPR/Cas9 system can be highly active even when mismatches occur between gRNA and their DNA targets”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [92]
In the citing paper
“and off-target mutations occur at endogenous loci in human cells with high frequency [90,91,92].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [92]
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Cites
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InfluentialRationally engineered Cas9 nucleases with improved specificity
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A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging
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InfluentialBoosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system
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BackgroundResultInfluentialAnalysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases
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BackgroundInfluentialCHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing
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“Cho et al. (2013) used the S. pyogenes Cas9 system (SpCas9) to edit human cells to incorporate GFP and RFP. Mali et al. (2013) engineered the SpCas9 system and cloned it into human cells, then performed multiplexed editing of target loci.”
BackgroundMethodInfluentialIn vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy
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MethodInfluentialCRISPR/Cas9-mediated gene editing in human tripronuclear zygotes
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BackgroundDigenome-seq: Genome-wide profiling of CRISPR-cas9 off-target effects in human cells
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“As noted above, an important weakness of the paired Cas9 nickase approach is that single monomeric nickases can introduce indel mutations with high frequencies at certain target sites 4, 10, 11, 13 .”
BackgroundApplications of CRISPR technologies in research and beyond
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CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering
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Principles of genetic circuit design
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BackgroundInfluentialCRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity
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BackgroundInfluentialDevelopment of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology
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“Each target sequence of 20 nucleotides was designed according to the criteria described previously (Mali et al., 2013) upstream of the NGG trinucleotide, known as the protospacer adjacent motif (PAM).”
MethodBeyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation
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“It has been suggested that a ‘‘dual nick’’ strategy, in which two gRNAs direct the Cas9D10A single strand nickase activity to nearby sites to yield an effective double strand break, will be a effective means to minimize off-target mutagenesis [11,13], because the two gRNAs will rarely yield nearby nicks outside of the…”
Adeno-associated virus as a delivery vector for gene therapy of human diseases
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Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects
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Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
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RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System
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BackgroundGenome-editing Technologies for Gene and Cell Therapy
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Metadata
- Authors
- Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M.
- Affiliations
- Harvard University (US)
- Corresponding author
- Church, George M
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2013-08-01
- Volume
- 31
- Issue
- 9
- Pages
- 833-838
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3818127
- OpenAlex
- W2026520402
- Cited by
- 1,752
- References
- 30
- Citations by year
- 2026: 33 · 2025: 45 · 2024: 71 · 2023: 83 · 2022: 122 · 2021: 119 · 2020: 125 · 2019: 159 · 2018: 154 · 2017: 190 · 2016: 231 · 2015: 175 · 2014: 209 · 2013: 36
- FWCI
- 97.27
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Human Genome Research Institute (P50HG005550)
- 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, transcriptional activation, single guide RNA, off-target binding, target specificity, TAL effectors, genome editing, target mismatch
- MeSH terms
- Base Sequence, Deoxyribonuclease I, Genetic Engineering, Humans, Models, Genetic, Molecular Sequence Data, Trans-Activators, HEK293 Cells, RNA, Small Untranslated, CRISPR-Associated Proteins
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