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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]
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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Analysis of epigenetic features characteristic of L1 loci expressed in human cells
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Gene targeting as a therapeutic avenue in diseases mediated by the complement alternative pathway
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Clinical genome editing to treat sickle cell disease—A brief update
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Context influences on TALE–DNA binding revealed by quantitative profiling
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A transcription activator–like effector (TALE) induction system mediated by proteolysis
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“SpCas9 has also been modified to completely remove its nuclease activity (termed dCas9 for ‘catalytically dead Cas9’) to allow for activator and repressor protein fusions [26,27].”
BackgroundInfluentialXie, C., Chen, Y., Wang, D., Wang, Y., Zhang, T., Li, H. Z., Fu, L., Zhao, Y. B., & Zhang, G. (2017) · Scientific Reports · doi:10.1038/s41598-017-06216-w · Cited by 33
“As to function, (1) the transcription of multiple sgRNAs and miRNAs can be triggered by a single promoter; (2) integration of diverse sgRNAs and miRNAs in one single vector makes it feasible to edit (or transcriptionally activate) and knock down a myriad of genes efficiently and simultaneously; (3) miRNA-based sgRNA e…”
BackgroundPrecise Cas9 targeting enables genomic mutation prevention
Chavez, A., Pruitt, B. W., Tuttle, M. L., Shapiro, R. S., Cecchi, R. J., Winston, J., Turczyk, B. M., Tung, M., Collins, J. J., & Church, G. M. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1718148115 · Cited by 32
Applications of CRISPR/Cas9 in the Mammalian Central Nervous System.
Savell, K. E., & Day, J. J. (2017) · PubMed · Cited by 33
Using CRISPR/Cas9 to model human liver disease
Alves‐Bezerra, M., Furey, N., Johnson, C. G., & Bissig, K. (2019) · JHEP Reports · doi:10.1016/j.jhepr.2019.09.002 · Cited by 32
Application of CRISPR genetic screens to investigate neurological diseases
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Park, H., & Kim, J. (2022) · Journal of Pineal Research · doi:10.1111/jpi.12787 · Cited by 33
“Among these dCas9 applications, an innovative technology called CRISPR/Cas9 activator, which overcomes the limitations of gene overexpression, has recently garnered increasing attention.(30,33) The Cas9 activator has been widely used to program the fate of pluripotent stem cells,(34) cardiac progenitor cells,(35) neur…”
BackgroundAllele-specific silencing of the gain-of-function mutation in Huntington’s disease using CRISPR/Cas9
Shin, J. W., Hong, E. P., Park, S. S., Choi, D. E., Seong, I. S., Whittaker, M. N., Kleinstiver, B. P., Chen, R. Z., & Lee, J. (2022) · JCI Insight · doi:10.1172/jci.insight.141042 · Cited by 37
“In support, challenges in genome editing in iPSC have been observed in the field (85-87).”
CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells
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CRISPR-Cas9 Genome Editing for Treatment of Atherogenic Dyslipidemia
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Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications
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Gene editing for dyslipidemias: New tools to “cut” lipids
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CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering
Glinšek, K., Bozovičar, K., & Bratkovič, T. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24098144 · Cited by 35
Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system
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Plant gene expression control using genome- and epigenome-editing technologies
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“The sgRNAs were modified by appending RNA aptamers (such as MS2, PP7, com or the PUF binding site), whose secondary RNA structures are explicitly recognized by RNA-binding proteins, to constitute a scaffolding RNA (scRNA) (Mali et al. 2013; Konermann et al. 2015).”
BackgroundThe CRISPR/Cas9 system for gene editing and its potential application in pain research
Sun, L., Marie, L. B., & Yuan-Xiang, T. (2016) · Translational Perioperative and Pain Medicine · doi:10.31480/2330-4871/040 · Cited by 32
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Genome editing in horticultural crops: augmenting trait development and stress resilience
Sohail, H., Noor, I., Hussain, H., Zhang, L., Xu, X., Chen, X., & Yang, X. (2025) · Horticultural Plant Journal · doi:10.1016/j.hpj.2025.09.001 · Cited by 34
Strategies for gene disruption in Drosophila
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“The system, which originated as a component of prokaryotic innate immunity system [55], recognizes target loci via sgRNA sequence: this 20-bp sgRNA requires an adjourning NGG sequence known as the protospacer adjacent motif (PAM) site for Cas9 recognition [56-58] (Figure 3C).”
BackgroundD-repeat in theXISTgene is required for X chromosome inactivation
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“chromatin structure.(16,17) In addition, off-target mutations occurred in D-repeat while not in POTSs in the human genome, which is not surprising because there are more mismatches in these 5 top POTSs than in the sgRNA targeted Drepeat sequence.”
BackgroundPrediction and diversity of tracrRNAs from type II CRISPR-Cas systems
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“In sgRNA constructs additional RNA loops or sequences have be added at the 3ʹ end of the tracrRNA [21,22] or by extending SL2 of S.”
BackgroundGene activation by a CRISPR-assisted trans enhancer
Xu, X., Gao, J., Dai, W., Wang, D., Wu, J., & Wang, J. (2019) · eLife · doi:10.7554/elife.45973 · Cited by 31
“In general, three or more sgRNAs are used to activate a gene of interest (Cheng et al., 2013; Maeder et al., 2013; Mali et al., 2013a; Perez-Pinera et al., 2013).”
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Current advances in overcoming obstacles of CRISPR/Cas9 off-target genome editing
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Protein Engineering of Cas9 for Enhanced Function
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The I-TevI Nuclease and Linker Domains Contribute to the Specificity of Monomeric TALENs
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Zebrafish Models of Epilepsy and Epileptic Seizures
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“CRISPR/Cas9 system has been widely adopted in several organisms (Mali et al., 2013).”
BackgroundUnexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in
Rezza, A., Jacquet, C., Pillouer, A. L., Lafarguette, F., Ruptier, C., Billandon, M., Isnard, P., Trouttet, S., Thiam, K., Fraichard, A., et al. (2019) · Scientific Reports · doi:10.1038/s41598-019-40181-w · Cited by 31
CRISPR‐Cas systems in oral microbiome: From immune defense to physiological regulation
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Gene Therapy for Inherited Retinal Degeneration
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“…of DNA, can produce DSBs with 2 times more specificity and 50–1,500 times less off-target effects compared with SpCas9 at the same time, 136,138,159,164,165 and (6) high-fidelity Cas9 (SpCas9-HF1) has 4 altered amino acids, compared with the wild-type SpCas9, with higher genomic editing efficiency…”
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Dissecting the Genetic Basis of a Complex cis-Regulatory Adaptation
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“Because AG PAM sequences were reported to work as well as GG PAM sequences with dCas9 activators in human cell lines [40], we tested gRNAs with both type of PAM.”
MethodThe Rise of CRISPR/Cas for Genome Editing in Stem Cells
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Energy biotechnology in the CRISPR-Cas9 era
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Genome Engineering with TALE and CRISPR Systems in Neuroscience
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“This bioinformatic identification of off-target sites was the basis of early studies in which these selected sites were monitored with reporter systems and sequenced (Cradick et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Mali et al., 2013a; Cho et al., 2014; Fu et al., 2014).”
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“It was reported that up to five mis-matches are tolerated by CAS9 in the gRNA sequence to potentially cut at hundreds of alternative sites across the genome (Fu et al., 2013; Mali et al., 2013).”
InfluentialCRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells
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Qiu, M., Glass, Z., & Xu, Q. (2019) · Biomacromolecules · doi:10.1021/acs.biomac.9b00783 · Cited by 30
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“SpCas9 nickases were generated to overcome the issue of off-target mutations that are commonly observed during genome editing (Mali et al. 2013).”
BackgroundSlattery, S. S., Wang, H. H., Giguere, D. J., Kocsis, C., Urquhart, B. L., Karas, B. J., & Edgell, D. R. (2020) · Scientific Reports · doi:10.1038/s41598-020-70769-6 · Cited by 30
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“Moreover, Cas9n, a variant of Cas9 with point mutation in the RuvC nuclease domain, can nick DNA but not cleave it [54,67].”
BackgrounddCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy
Riedmayr, L. M., Hinrichsmeyer, K. S., Karguth, N., Böhm, S., Splith, V., Michalakis, S., & Bećirović, E. (2022) · Nature Protocols · doi:10.1038/s41596-021-00666-3 · Cited by 33
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“119,121 Targeting the Gal4 upstream activation sequence (UAS), these sgRNA-bound fusion proteins were concurrently transfected into HEK-293 T cells, which expressed a green fluorescent protein (GFP).”
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CRISPR Tools for Systematic Studies of RNA RegulationPreprint
Engreitz, J., Abudayyeh, O. O., Gootenberg, J. S., & Zhang, F. (2019) · Cold Spring Harbor Perspectives in Biology · doi:10.1101/cshperspect.a035386 · Cited by 31
“…Cite this article as Cold Spring Harb Perspect Biol 2019;11:a035386 of gene expression at promoters (Bikard et al. 2013; Cheng et al. 2013; Farzadfard et al. 2013; Gilbert et al. 2013, 2014; Maeder et al. 2013; Mali et al. 2013a; Perez-Pinera et al. 2013; Chavez et al. 2015; Konermann et al. 2015).”
BackgroundInfluentialAn Overview of Genome‐Engineering Methods
Sushmita, S., Kaur, G., Upadhyay, S. K., & Verma, P. C. (2021) · doi:10.1002/9781119672425.ch1 · Cited by 29
Karpov, D. S., Sosnovtseva, A. O., Pylina, S. V., Bastrich, A., Petrova, D. A., Kovalev, M. A., Shuvalova, A. I., Константиновна, Е. А., & Mokrysheva, N. G. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242417320 · Cited by 30
Weber, N. D., Aubert, M., Dang, C. H., Stone, D., & Jerome, K. R. (2014) · Virology · doi:10.1016/j.virol.2013.12.037 · Cited by 28
Sequence-specific DNA nicking endonucleases
Xu, S.-Y. (2015) · BioMolecular Concepts · doi:10.1515/bmc-2015-0016 · Cited by 28
Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology
Wu, J., Hunt, S. D., Xue, H., Liu, Y., & Darabi, R. (2016) · Stem Cell Research · doi:10.1016/j.scr.2016.01.003 · Cited by 28
Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease
Powell, S. K., Gregory, J. K., Akbarian, S., & Brennand, K. (2017) · Molecular and Cellular Neuroscience · doi:10.1016/j.mcn.2017.05.007 · Cited by 30
“Mutation of one of the two Cas9 nuclease domains creates a “nickase Cas9 (nCas9)” that cleaves only one of the target DNA strands (Mali et al., 2013), and thus enhances target specificity, as only adjacent nCas9-mediated cuts will bring about the desired DSB (Mali et al.”
InfluentialRusso, M. T., Cigliano, R. A., Sanseverino, W., & Ferrante, M. I. (2018) · PeerJ · doi:10.7717/peerj.5507 · Cited by 26
“Off-target mutations in human cells were reported by some authors to be more common than mutations at the on-target site, raising concerns about the intrinsic fidelity of the CRISPR/Cas9 system (Fu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013; Hsu, Lander & Zhang, 2014; Kim et al., 2015).”
BackgroundScharf, A., Lang, C., & Fischer, M. J. (2020) · Food Control · doi:10.1016/j.foodcont.2020.107219 · Cited by 28
ROLE OF CRISPR TO IMPROVE ABIOTIC STRESS TOLERANCE IN CROP PLANTS
Farooq, M., Bashir, M., Khan, M., Iqbal, B., & Ali, Q. (2021) · Biological and Clinical Sciences Research Journal · doi:10.54112/bcsrj.v2021i1.69 · Cited by 26
“Genome modified crops have such a benefit over transgenic crops in that they ‘carry' the target trait's edited DNA (Mali et al., 2013).”
BackgroundEisenhut, P., Marx, N., Borsi, G., Papež, M., Ruggeri, C., Baumann, M., & Borth, N. (2023) · New Biotechnology · doi:10.1016/j.nbt.2023.11.003 · Cited by 33
Targeted genome regulation via synthetic programmable transcriptional regulators
Piatek, A., & Mahfouz, M. M. (2016) · Critical Reviews in Biotechnology · doi:10.3109/07388551.2016.1165180 · Cited by 28
Targeting cellular mRNAs translation by CRISPR-Cas9
Liu, Y., Chen, Z., He, A., Zhan, Y., Li, J., Liu, L., Wu, H., Zhuang, C., Lin, J., Zhang, Q., et al. (2016) · Scientific Reports · doi:10.1038/srep29652 · Cited by 28
Gene editing prospects for treating inherited retinal diseases
Benati, D., Patrizi, C., & Recchia, A. (2019) · Journal of Medical Genetics · doi:10.1136/jmedgenet-2019-106473 · Cited by 28
Wilkinson, A. C., Kawata-Matsuura, V. K. S., Schütte, J., Gao, X., Antoniou, S., Baumann, C., Woodhouse, S., Hannah, R., Tanaka, Y., Swiers, G., et al. (2014) · Development · doi:10.1242/dev.115709 · Cited by 27
“However, a recent comparison between CRISPR-Cas9 and TALEs D E V E LO P M E N T suggested higher targeting specificity for the latter (Fu et al., 2013; Mali et al., 2013).”
Insights into neural crest development from studies of avian embryos
Gandhi, S., & Bronner-Fraser, M. E. (2018) · The International Journal of Developmental Biology · doi:10.1387/ijdb.180038sg · Cited by 26
CRISPR-Cas9 in agriculture: Approaches, applications, future perspectives, and associated challenges
Adhikari, P., & Poudel, M. (2020) · Malaysian Journal of Halal Research · doi:10.2478/mjhr-2020-0002 · Cited by 23
“Modification of cas9 by the cas9 nuclease inactivation and double nicking using nickase enhances the specificity[37].”
MethodA primer to gene therapy: Progress, prospects, and problems
Zittersteijn, H. A., Gonçalves, M. A. F. V., & Hoeben, R. C. (2020) · Journal of Inherited Metabolic Disease · doi:10.1002/jimd.12270 · Cited by 28
“Paired chromosomal SSBs formed by two nicking CRISPR-Cas9 complexes with gRNAs targeting sequences on opposite DNA strands located near to each other, can generate a targeted DSB.(150,151) Crucially, with this dual CRISPR-Cas9 nickase strategy, off-target activities are greatly reduced in that SSBs made elsewhere in t…”
BackgroundInfluentialTranscriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing
Moses, C., Hodgetts, S. I., Nugent, F., Ben-Ary, G., Park, K. K., Blancafort, P., & Harvey, A. R. (2020) · Scientific Reports · doi:10.1038/s41598-020-68257-y · Cited by 28
Creating Cancer Translocations in Human Cells Using Cas9 DSBs and nCas9 Paired Nicks
Renouf, B., Piganeau, M., Ghezraoui, H., Jasin, M., & Brunet, E. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00012-x · Cited by 30
“When two gRNAs are provided which bind opposite DNA strands in close proximity, however, paired nicks can be introduced, also creating a DSB but with a long overhang (Mali et al., 2013b; Ran, Hsu, Lin, Methods Enzymol.”
BackgroundHIT-Cas9: A CRISPR/Cas9 Genome-Editing Device under Tight and Effective Drug Control
Zhao, C., Zhao, Y., Zhang, J., Lu, J. Q., Chen, L., Zhang, Y., Yue, Y., Xu, J., Wei, S., & Wang, Y. (2018) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2018.08.022 · Cited by 27
“In addition, mCherry signal in the TLR reporter that probes NHEJ events displayed a high background noise, consistent with its previous use after prior purification to remove mCherry-positive cells.(26) Moreover, CD201 knockout assay, although quite sensitive in detection of NHEJ events, required prolonged culture for…”
Choi, J., Bae, T., Byambasuren, N., Park, S.-H., Jo, C. H., Kim, D., Hur, J. K., & Hwang, N. S.-Y. (2020) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2019.12.010 · Cited by 27
Therapeutic genome engineering via CRISPR ‐Cas systems
Moreno, A. M., & Mali, P. (2017) · WIREs Systems Biology and Medicine · doi:10.1002/wsbm.1380 · Cited by 25
“Studies on the specificity of SpCas9 have demonstrated that mismatches between gRNA and target DNA can be tolerated.(43,57,89,95) Moreover, the farther mismatches are from the PAM sequence, the more tolerated that they are.”
BackgroundApplications of CRISPR-Cas for synthetic biology and genetic recording
Schmidt, F., & Platt, R. J. (2017) · Current Opinion in Systems Biology · doi:10.1016/j.coisb.2017.05.008 · Cited by 26
Das, A., Hand, T. H., Smith, C. L., Wickline, E., Zawrotny, M., & Li, H. (2020) · Nature Communications · doi:10.1038/s41467-020-20204-1 · Cited by 25
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Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1
Torella, L., Klermund, J., Bilbao‐Arribas, M., Tamayo, I., Andrieux, G., Chmielewski, K. O., Vales, Á., Olagüe, C., Moreno, D., Raimondi, I., et al. (2024) · EMBO Molecular Medicine · doi:10.1038/s44321-023-00008-8 · Cited by 27
“However, simultaneous nicking of both DNA strands using paired nickases that are appropriately spaced and oriented leads to site-speci fi c staggered DSB formation (Ran et al, 2013a; Mali et al, 2013a).”
Fish genome manipulation and directional breeding
Ding, Y., Zhu, Z., & Sun, Y. (2015) · Science China Life Sciences · doi:10.1007/s11427-015-4806-7 · Cited by 26
Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing
Kaulich, M., & Dowdy, S. F. (2015) · Nucleic Acid Therapeutics · doi:10.1089/nat.2015.0545 · Cited by 25
Hou, S., Qin, Q., & Dai, J. (2018) · ACS Synthetic Biology · doi:10.1021/acssynbio.7b00391 · Cited by 26
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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