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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TALENs can tolerate two mismatches in vivo.
“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]
CRISPR/Cas9 system can be highly active even when mismatches occur between gRNA and their DNA targets.
“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]
Off-target mutations occur at endogenous loci in human cells with high frequency.
“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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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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Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian 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
Barrangou, R., & Doudna, J. A. (2016) · Nature Biotechnology · doi:10.1038/nbt.3659 · Cited by 1,005 · see citing works
CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering
Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B., & Valen, E. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw398 · Cited by 910 · see citing works
Principles of genetic circuit design
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Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds
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Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila
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“…been shown that the CRISPR/Cas9 complex can tolerate mismatches particularly at the 59 end of the targeting sequence (Semenova et al. 2011; Wiedenheft et al. 2011; Cradick et al. 2013; Fu et al. 2013; Hsu et al. 2013; Mali et al. 2013b; Pattanayak et al. 2013; Yang et al. 2013; Cho et al. 2014).”
BackgroundInfluentialCRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity
Barrangou, R., & Marraffini, L. A. (2014) · Molecular Cell · doi:10.1016/j.molcel.2014.03.011 · Cited by 907 · see citing works
“In mammalian cells, dCas9 can be fused to transcriptional activators such as VP64 and the p65 activator domain (Figure 3) (Gilbert et al., 2013; Maeder et al., 2013; Mali et al., 2013a).”
BackgroundInfluentialDevelopment of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology
Chandrasekaran, J., Brumin, M., Wolf, D., Leibman, D., Klap, C., Pearlsman, M., Sherman, A., Arazi, T., & Gal‐On, A. (2016) · Molecular Plant Pathology · doi:10.1111/mpp.12375 · Cited by 883 · see citing works
“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
Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm.2015.2 · Cited by 832 · see citing works
Concerning RNA-guided gene drives for the alteration of wild populations
Esvelt, K. M., Smidler, A. L., Catteruccia, F., & Church, G. M. (2014) · eLife · doi:10.7554/elife.03401 · Cited by 857 · see citing works
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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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Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J., & Church, G. M. (2013) · Nature Methods · doi:10.1038/nmeth.2681 · Cited by 786 · see citing works
CRISPR-mediated direct mutation of cancer genes in the mouse liver
Xue, W., Chen, S., Yin, H., Tammela, T., Papagiannakopoulos, T., Joshi, N. S., Cai, W., Yang, G., Bronson, R., Crowley, D. G., et al. (2014) · Nature · doi:10.1038/nature13589 · Cited by 776 · see citing works
“We next tested a nickase version of Cas9, Cas9 D10A , which only makes single strand DNA breaks and was reported to have further reduced levels of off-target effects 23, 24 .”
DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins
Malnoy, M., Viola, R., Jung, M., Koo, O. J., Kim, S., Kim, J., Velasco, R., & Kanchiswamy, C. N. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.01904 · Cited by 766 · see citing works
“Synthesis of unique sgRNA (Cho et al., 2014; Fu et al., 2014; Sung et al., 2014), Cas9 nickases and web based computer programs to identify unique target sites (Mali et al., 2013; Ran et al., 2013; Bae et al., 2014) and CRISPR/Cas9 RNPs (Woo et al., 2015).”
BackgroundGenome-editing Technologies for Gene and Cell Therapy
Maeder, M. L., & Gersbach, C. A. (2016) · Molecular Therapy · doi:10.1038/mt.2016.10 · Cited by 722 · see citing works
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A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
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“Most recently, the Streptococcus pyogenes CRISPR (clustered regularly interspaced 118 short palindromic repeats)/Cas9 system has burst on the scene, after it was shown to effectively 119 mediate RNA-guided DNA double strand breaks in bacteria and mammalian cells (Jinek et al., 120 2012; Cong et al., 2013; Mali et al.,…”
BackgroundInfluentialA CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
Nødvig, C. S., Nielsen, J. B., Kogle, M. E., & Mortensen, U. H. (2015) · PLoS ONE · doi:10.1371/journal.pone.0133085 · Cited by 701 · see citing works
Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
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CRISPR/Cas9 for genome editing: progress, implications and challenges
Zhang, F., Wen, Y., & Guo, X. (2014) · Human Molecular Genetics · doi:10.1093/hmg/ddu125 · Cited by 694 · see citing works
CRISPR for Crop Improvement: An Update Review
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Lin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., Wile, B. M., Vertino, P. M., Stewart, F. J., & Bao, G. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku402 · Cited by 634 · see citing works
Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy
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A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants
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“Consistent with the data from human cells (Mali et al., 2013; Ran et al., 2013), paired nickases that create 59 overhangs induced GT more frequently than those creating 39 overhangs.”
BackgroundA light-inducible CRISPR-Cas9 system for control of endogenous gene activation
Polstein, L. R., & Gersbach, C. A. (2015) · Nature Chemical Biology · doi:10.1038/nchembio.1753 · Cited by 630 · see citing works
Photoactivatable CRISPR-Cas9 for optogenetic genome editing
Nihongaki, Y., Kawano, F., Nakajima, T., & Sato, M. (2015) · Nature Biotechnology · doi:10.1038/nbt.3245 · Cited by 609 · see citing works
Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications
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“Lipid nanoparticles have been explored to deliver the CRISPR-Cas9 systems to numerous cells [7,47] for therapeutic purpose or to establish knockout animal models [76,77].”
BackgroundEpigenetics and epigenomics in diabetic kidney disease and metabolic memory
Kato, M. V., & Natarajan, R. D. (2019) · Nature Reviews Nephrology · doi:10.1038/s41581-019-0135-6 · Cited by 604 · see citing works
Delivery technologies for genome editing
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Genome Engineering with Targetable Nucleases
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“Three groups (99, 101, 179) describe an alternative that makes the CRISPR system more specific.”
BackgroundMethodExpanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9
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Comparison of Cas9 activators in multiple species
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Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR
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Genome Engineering Using CRISPR-Cas9 System
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Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases
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Classification and evolution of type II CRISPR-Cas systems
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A highly specific SpCas9 variant is identified by in vivo screening in yeast
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“…been demonstrated to produce off-target effects, i.e., mutagenesis at sites in the genome other than the desired on-target site (Hockemeyer et al., 2011; Mussolino et al., 2011; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013; Cradick et al., 2013; Cho et al., 2014).”
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“Furthermore, improvements to the specificity of the CRISPR/Cas9 system, including the use of dual nickases, truncated sgRNAs, fusions to a FokI endonuclease domain, and careful sgRNA selection methods 15, 18, 19, 44–46 , are also under development.”
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Editing the epigenome: technologies for programmable transcription and epigenetic modulation
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- 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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