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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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In the citing paper
“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]
In the citing paper
“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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“…induce repair by NHEJ or HDR. Off-target mutagenesis is substantially reduced with the double-nickase strategy because the two nicks occur in proximity only when Cas9 binds to two adjacent sequences that resemble the protospacers, which is very unlikely to occur elsewhere in the genome (75–77).”
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“Inspired by this discovery, both of FokI [40, 48] and Cas9 [23, 31, 43, 44, 49] have been modified to act as a nickase to stimulate HDR.”
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Functional interrogation of non-coding DNA through CRISPR genome editing
Canver, M. C., Bauer, D. E., & Orkin, S. H. (2017) · Methods · doi:10.1016/j.ymeth.2017.03.008 · Cited by 36
One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes
Li, S., Zhang, A., Xue, H., Li, D., & Liu, Y. (2017) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2017.06.007 · Cited by 35
CRISPR Genome Engineering for Human Pluripotent Stem Cell Research
Chaterji, S., Ahn, E. H., & Kim, D. (2017) · Theranostics · doi:10.7150/thno.18456 · Cited by 35
“Notably, most nicks result in very low indel rates resulting in effective targeted gene disruption [124, 125].”
BackgroundSplit Cas9, Not Hairs − Advancing the Therapeutic Index of CRISPR Technology
Schmelas, C., & Grimm, D. (2018) · Biotechnology Journal · doi:10.1002/biot.201700432 · Cited by 35
Chen, X., Tasca, F., Wang, Q., Liu, J., Janssen, J. M., Brescia, M., Bellin, M., Szuhai, K., Kenrick, J., Frock, R. L., et al. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz1121 · Cited by 37
Delivery Platforms for CRISPR/Cas9 Genome Editing of Glial Cells in the Central Nervous System
Meneghini, V., Peviani, M., Luciani, M., Zambonini, G., & Gritti, A. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.644319 · Cited by 36
“Increased epigenetic activity has been achieved by fusing the dCas9 with four tandem V16 copies leading to the generation of the epigenetic activator dCas9–VP64 (Maeder et al., 2013; Mali et al., 2013; Perez-Pinera et al., 2013).”
BackgroundCRISPR/Cas9 a simple, inexpensive and effective technique for gene editing
Ferreira, P., & Choupina, A. (2022) · Molecular Biology Reports · doi:10.1007/s11033-022-07442-w · Cited by 39
Vriend, L. E. M., Jasin, M., & Krawczyk, P. M. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00009-x · Cited by 35
“…bacterial adaptive immunity system, CRISPR/Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated), has enabled straightforward induction of DSBs and SSBs at desired loci by the RNA-guided Cas9 endonuclease (Gasiunas et al., 2012; Jinek et al., 2012; Mali et al., 2013a).”
BackgroundCRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering
Singh, A., Chakraborty, D., & Maiti, S. (2016) · Chemical Society Reviews · doi:10.1039/c6cs00197a · Cited by 35
Safety, Security, and Policy Considerations for Plant Genome Editing
Wolt, J. D. (2017) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2017.03.005 · Cited by 37
Modularized CRISPR/dCas9 Effector Toolkit for Target-Specific Gene Regulation
Agne, M., Blank, I., Emhardt, A. J., Gäbelein, C. G., Gawlas, F., Gillich, N., Gonschorek, P., Juretschke, T., Krämer, S., Louis, N., et al. (2014) · ACS Synthetic Biology · doi:10.1021/sb500035y · Cited by 35
The genome editing toolbox: a spectrum of approaches for targeted modification
Cheng, J. K., & Alper, H. S. (2014) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2014.06.005 · Cited by 34
Vaughn, J. N., & Bennetzen, J. L. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1321854111 · Cited by 34
Rathe, S. K., Moriarity, B. S., Stoltenberg, C. B., Kurata, M., Aumann, N. K., Rahrmann, E. P., Bailey, N. J., Melrose, E. G., Beckmann, D. A., Liska, C. R., et al. (2014) · Scientific Reports · doi:10.1038/srep06048 · Cited by 34
Nanomedicine: Tiny Particles and Machines Give Huge Gains
Tong, S., Fine, E. J., Lin, Y., Cradick, T. J., & Bao, G. (2013) · Annals of Biomedical Engineering · doi:10.1007/s10439-013-0952-x · Cited by 34
Multi-gene engineering in plants with RNA-guided Cas9 nuclease
Raitskin, O., & Patron, N. J. (2015) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2015.11.008 · Cited by 38
Advances and perspectives in the application of CRISPR/Cas9 in insects.
Chen, L., Wang, G., Zhu, Y., Xiang, H., & Wang, W. (2016) · PubMed · doi:10.13918/j.issn.2095-8137.2016.4.220 · Cited by 34
“Cas9 nickase (dCas9) with paired gRNAs properly positioned on the target DNA exhibits low off-target mutagenesis compared with wild-type Cas9 (Cho et al., 2014; Mali et al., 2013; Ran et al., 2013).”
BackgroundMethodHarnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox
Karvelis, T., Gasiūnas, G., & Šikšnys, V. (2017) · Current Opinion in Microbiology · doi:10.1016/j.mib.2017.05.009 · Cited by 33
CRISPR/Cas‐mediated gene editing using purified protein in D rosophila suzukii
Kalajdzic, P., & Schetelig, M. F. (2017) · Entomologia Experimentalis et Applicata · doi:10.1111/eea.12599 · Cited by 34
“Additionally, Cas9/gRNA cleavage data showed that a G to A mismatch (NAG instead of NGG) in the PAM sequence could lead to off-targeting cleavage activity (Hsu et al., 2013; Mali et al., 2013).”
BackgroundApplications of the CRISPR/Cas system beyond gene editing
Anton, T., Karg, E., & Bultmann, S. (2018) · Biology Methods and Protocols · doi:10.1093/biomethods/bpy002 · Cited by 32
“Combining this modified sgRNA with a dCas9-VP64 fusion resulted in drastically increased expression levels of targeted genes and was shown to simultaneously upregulate 10 genes [114].”
BackgroundDNA, RNA, and Protein Tools for Editing the Genetic Information in Human Cells
Chen, X., & Gonçalves, M. A. F. V. (2018) · iScience · doi:10.1016/j.isci.2018.08.001 · Cited by 36
CRISPR/Cas9-mediated mutagenesis of homologous genes in Chinese kale
Sun, B., Zheng, A., Jiang, M., Xue, S., Yuan, Q., Jiang, L., Chen, Q., Li, M., Wang, Y., Zhang, Y., et al. (2018) · Scientific Reports · doi:10.1038/s41598-018-34884-9 · Cited by 34
A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast
Hofmann, A., Falk, J., Prangemeier, T., Happel, D., Köber, A., Christmann, A., Koeppl, H., & Kolmar, H. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky1191 · Cited by 36
CRISPR-RT: a web application for designing CRISPR-C2c2 crRNA with improved target specificity
Zhu, H., Richmond, E., & Liang, C. (2017) · Bioinformatics · doi:10.1093/bioinformatics/btx580 · Cited by 34
Mollanoori, H., Rahmati, Y., Hassani, B., Havasi Mehr, M., & Teimourian, S. (2021) · Genes & Diseases · doi:10.1016/j.gendis.2019.12.007 · Cited by 32
“The transcription activator domain operates as a recruiter for the multiple parts of the pre-initiation complex.(62,63) CRISPRa platform provides a novel opportunity for disease treatment by modulating the expression of genes that contribute to the pathogenesis of the disease.”
BackgroundGenome oligopaint via local denaturation fluorescence in situ hybridization
Wang, Y., Cottle, W. T., Wang, H., Feng, X. A., Mallon, J. P., Gavrilov, M. M., Bailey, S., & Ha, T. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.02.011 · Cited by 36
“CRISPR-mediated transcriptional activation and repression platforms (CRISPRa and CRISPRi) were developed utilizing dCas9 (Gilbert et al., 2013; Maeder et al., 2013; Mali et al., 2013a; Perez-Pinera et al., 2013; Qi et al., 2013).”
Method
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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