Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
Open accessPublished version of a preprint
Abstract
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Among the 134 off-target sites of one sgRNA targeting VEGFA with SpCas9, only 19, 24, and 18 of the off-target sites are also detectable with eSpCas9, SpCas9-HF1 and hypaCas9, respectively.
“For example, among the 134 off-target sites of one sgRNA targeting VEGFA with SpCas9, only 19, 24, and 18 of the off-target sites are also detectable with eSpCas9, SpCas9-HF1 and hypaCas9, respectively (Chen et al., 2017).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Chen et al., 2017
The paper’s genome-wide specificity passage (indexed under Abstract; page not provided) reports GUIDE-seq comparisons for VEGFA-targeting sgRNAs and improved specificity for the variants relative to wild-type SpCas9. The Extended Data Figure 9 caption describes off-target profiles, but our copy does not state the claimed counts of 134, 19, 24, and 18 or establish that those sites overlap. (LLM, conf=0.78)
Experimental — accuracy not yet measuredhypaCas9 demonstrates higher on-target activity and lower off-target effects than eSpCas9 and SpCas9-HF1.
“Accordingly, scientists further designed hypaCas9 (hyper-accurate Cas9) (Chen et al., 2017), which demonstrates higher on-target activity and lower off-target effects than eSpCas9 and SpCas9-HF1 (Kleinstiver et al., 2016; Slaymaker et al., 2016; Chen et al., 2017).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Chen et al., 2017
The GUIDE-seq passage (section labeled Abstract; page not provided) reports comparable on-target editing efficiencies among HypaCas9, eSpCas9(1.1), and SpCas9-HF1—not higher activity for HypaCas9. It reports equivalent or better genome-wide specificity for HypaCas9, which supports a possible reduction in off-target effects but not a reduction relative to both variants for every sgRNA examined. (LLM, conf=0.90)
Experimental — accuracy not yet measuredeSpCas9, SpCas9-HF1 and hypaCas9 maintained high on-target activity (>70% of wildtype SpCas9) at 23/24, 18/24, and 19/24 tested sites, respectively.
“Janice S et al. shows that eSpCas9, SpCas9-HF1 and hypaCas9 maintained high on-target activity (>70% of wildtype SpCas9) at 23/24, 18/24, and 19/24 tested sites, respectively.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Chen et al., 2017
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Self-citationThe new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062 · see citing works
Self-citationDevelopment and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762 · see citing works
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · see citing works
Self-citationHigh-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016) · Nature · doi:10.1038/nature16526 · Cited by 2,673 · see citing works
Self-citationRationally engineered Cas9 nucleases with improved specificity
Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., & Zhang, F. (2016) · Science · doi:10.1126/science.aad5227 · Cited by 2,414 · see citing works
Bae, S., Park, J., & Kim, J.-S. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu048 · Cited by 2,339 · see citing works
Crystal structure of Cas9 in complex with guide RNA and target DNA
Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014) · Cell · doi:10.1016/j.cell.2014.02.001 · Cited by 2,306 · see citing works
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268 · see citing works
Self-citationEngineered CRISPR-Cas9 nucleases with altered PAM specificities
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P. W., Li, Z., Peterson, R. T., Yeh, J.-R. J., et al. (2015) · Nature · doi:10.1038/nature14592 · Cited by 1,624 · see citing works
Self-citationStructural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease
Anders, C., Niewoehner, O., Duerst, A., & Jínek, M. (2014) · Nature · doi:10.1038/nature13579 · Cited by 1,319 · see citing works
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
Jinek, M., Jiang, F., Taylor, D. W., Sternberg, S. H., Kaya, E., Ma, E., Anders, C., Hauer, M., Zhou, K., Lin, S., et al. (2014) · Science · doi:10.1126/science.1247997 · Cited by 1,230 · see citing works
Self-citationCas9 as a versatile tool for engineering biology
Mali, P., Esvelt, K. M., & Church, G. M. (2013) · Nature Methods · doi:10.1038/nmeth.2649 · Cited by 1,197 · see citing works
FLASH assembly of TALENs for high-throughput genome editing
Reyon, D., Tsai, S. Q., Khayter, C., Foden, J. A., Sander, J. D., & Joung, J. K. (2012) · Nature Biotechnology · doi:10.1038/nbt.2170 · Cited by 1,171 · see citing works
Self-citationLin, 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
Conformational control of DNA target cleavage by CRISPR–Cas9
Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015) · Nature · doi:10.1038/nature15544 · Cited by 627 · see citing works
Self-citationStructures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
Jiang, F., Taylor, D. W., Chen, J. S., Kornfeld, J. E., Zhou, K., Thompson, A. J., Nogales, E., & Doudna, J. A. (2016) · Science · doi:10.1126/science.aad8282 · Cited by 616 · see citing works
Self-citationA Cas9–guide RNA complex preorganized for target DNA recognition
Jiang, F., Zhou, K., Ma, L., Gressel, S., & Doudna, J. A. (2015) · Science · doi:10.1126/science.aab1452 · Cited by 551 · see citing works
Self-citationDefining and improving the genome-wide specificities of CRISPR–Cas9 nucleases
Tsai, S. Q., & Joung, J. K. (2016) · Nature Reviews Genetics · doi:10.1038/nrg.2016.28 · Cited by 508 · see citing works
Self-citationDirect observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes
Szczelkun, M. D., Tikhomirova, M. S., Šinkūnas, T., Gasiūnas, G., Karvelis, T., Pschera, P., Šikšnys, V., & Seidel, R. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1402597111 · Cited by 445 · see citing works
A decade of discovery: CRISPR functions and applications
Barrangou, R., & Horvath, P. (2017) · Nature Microbiology · doi:10.1038/nmicrobiol.2017.92 · Cited by 314 · see citing works
A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9
Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A., & Yildiz, A. (2017) · Science Advances · doi:10.1126/sciadv.aao0027 · Cited by 293 · see citing works
Self-citationRational design of a split-Cas9 enzyme complex
Wright, A. V., Sternberg, S. H., Taylor, D. W., Staahl, B. T., Bardales, J. A., Kornfeld, J. E., & Doudna, J. A. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1501698112 · Cited by 279 · see citing works
Self-citationCRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations
Palermo, G., Miao, Y., Walker, R. C., Jínek, M., & McCammon, J. A. (2017) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1707645114 · Cited by 177 · see citing works
Palermo, G., Miao, Y., Walker, R. C., Jínek, M., & McCammon, J. A. (2016) · ACS Central Science · doi:10.1021/acscentsci.6b00218 · Cited by 137 · see citing works
Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage
Cencic, R., Miura, H., Malina, A., Robert, F., Ethier, S., Schmeing, T. M., Dostie, J., & Pelletier, J. (2014) · PLoS ONE · doi:10.1371/journal.pone.0109213 · Cited by 117 · see citing works
Majumdar, Z. K., Hickerson, R. P., Noller, H. F., & Clegg, R. M. (2005) · Journal of Molecular Biology · doi:10.1016/j.jmb.2005.06.027 · Cited by 80 · see citing works
Open-source guideseq software for analysis of GUIDE-seq data
Tsai, S. Q., Topkar, V. V., Joung, J. K., & Aryee, M. J. (2016) · Nature Biotechnology · doi:10.1038/nbt.3534 · Cited by 78 · see citing works
Self-citationBisaria, N., Jarmoskaite, I., & Herschlag, D. (2017) · Cell Systems · doi:10.1016/j.cels.2016.12.010 · Cited by 72 · see citing works
Flagged first, then by citations
Research works printed but not linked: 1
- [30]Lin Y CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences Nucleic Acids Res 42 7473 7485 2014 24838573 PMC4066799
Cited by
Sorted by citations
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
Hu, J. H., Miller, S. M., Geurts, M. H., Tang, W., Chen, L., Sun, N., Zeina, C. M., Gao, X., Rees, H. A., Lin, Z., et al. (2018) · Nature · doi:10.1038/nature26155 · Cited by 1,552 · see citing works
CRISPR-Cas guides the future of genetic engineering
Knott, G. J., & Doudna, J. A. (2018) · Science · doi:10.1126/science.aat5011 · Cited by 1,410 · see citing works
Self-citationDunbar, C. E., High, K. A., Joung, J. K., Kohn, D. B., Ozawa, K., & Sadelain, M. (2018) · Science · doi:10.1126/science.aan4672 · Cited by 1,410 · see citing works
Self-citationVakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S., Camarena, J., et al. (2018) · Nature Medicine · doi:10.1038/s41591-018-0137-0 · Cited by 782 · see citing works
Off-target effects in CRISPR/Cas9 gene editing
Guo, C., Ma, X., Gao, F., & Guo, Y. (2023) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1143157 · Cited by 625 · see citing works
“For example, among the 134 off-target sites of one sgRNA targeting VEGFA with SpCas9, only 19, 24, and 18 of the off-target sites are also detectable with eSpCas9, SpCas9-HF1 and hypaCas9, respectively (Chen et al., 2017).”
BackgroundPast, present, and future of CRISPR genome editing technologies
Pacesa, M., Pelea, O., & Jinek, M. (2024) · Cell · doi:10.1016/j.cell.2024.01.042 · Cited by 609 · see citing works
Machine learning in healthcare
Habehh, H., & Gohel, S. (2021) · Current Genomics · doi:10.2174/1389202922666210705124359 · Cited by 544 · see citing works
Directed evolution of CRISPR-Cas9 to increase its specificity
Lee, J. K., Jeong, E., Lee, J., Jung, M., Shin, E., Kim, Y.-H., Lee, K., Jung, I., Kim, D., Kim, S., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-05477-x · Cited by 525 · see citing works
A highly specific SpCas9 variant is identified by in vivo screening in yeast
Casini, A., Olivieri, M., Petris, G., Montagna, C., Reginato, G., Maule, G., Lorenzin, F., Prandi, D., Romanel, A., Demichelis, F., et al. (2018) · Nature Biotechnology · doi:10.1038/nbt.4066 · Cited by 493 · see citing works
CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations
Cullot, G., Boutin, J., Toutain, J., Prat, F., Pennamen, P., Rooryck, C., Teichmann, M., Rousseau, E., Lamrissi-Garcia, I., Guyonnet-Duperat, V., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-09006-2 · Cited by 474 · see citing works
High levels of AAV vector integration into CRISPR-induced DNA breaks
Hanlon, K. S., Kleinstiver, B. P., Garcia, S. P., Zaborowski, M. P., Volak, A., Spirig, S. E., Muller, A., Sousa, A. A., Tsai, S. Q., Bengtsson, N. E., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-12449-2 · Cited by 431 · see citing works
Self-citationLatest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020) · Cells · doi:10.3390/cells9071608 · Cited by 457 · see citing works
DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing
Xue, C., & Greene, E. C. (2021) · Trends in Genetics · doi:10.1016/j.tig.2021.02.008 · Cited by 431 · see citing works
Precise plant genome editing using base editors and prime editors
Molla, K. A., Sretenovic, S., Bansal, K. C., & Qi, Y. (2021) · Nature Plants · doi:10.1038/s41477-021-00991-1 · Cited by 387 · see citing works
High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population
Wagner, D. L., Amini, L., Wendering, D. J., Burkhardt, L.-M., Akyüz, L., Reinke, P., Volk, H.-D., & Schmueck-Henneresse, M. (2019) · Nature Medicine · doi:10.1038/s41591-018-0204-6 · Cited by 399 · see citing works
CRISPR/Cas: A powerful tool for gene function study and crop improvement
Zhang, D., Zhang, Z., Unver, T., & Zhang, B. (2021) · Journal of Advanced Research · doi:10.1016/j.jare.2020.10.003 · Cited by 373 · see citing works
Increasing the specificity of CRISPR systems with engineered RNA secondary structures
Kocak, D. D., Josephs, E. A., Bhandarkar, V., Adkar, S. S., Kwon, J. B., & Gersbach, C. A. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0095-1 · Cited by 351 · see citing works
Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning
Wang, D., Zhang, C., Wang, B., Li, B., Wang, Q., Liu, D., Wang, H., Zhou, Y., Shi, L., Lan, F., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-12281-8 · Cited by 331 · see citing works
Gene therapy for neurological disorders: progress and prospects
Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M., & Sah, D. W. Y. (2018) · Nature Reviews Drug Discovery · doi:10.1038/nrd.2018.110 · Cited by 306 · see citing works
Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs
Ryan, D. E., Taussig, D., Steinfeld, I., Phadnis, S. M., Lunstad, B. D., Singh, M., Vuong, X., Okochi, K. D., McCaffrey, R., Olesiak, M., et al. (2018) · Nucleic Acids Research · doi:10.1093/nar/gkx1199 · Cited by 280 · see citing works
A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing
Edraki, A., Mir, A., Ibraheim, R., Gainetdinov, I., Yoon, Y., Song, C.-Q., Cao, Y., Gallant, J., Xue, W., Rivera-Pérez, J. A., et al. (2019) · Molecular Cell · doi:10.1016/j.molcel.2018.12.003 · Cited by 265 · see citing works
Partial DNA-guided Cas9 enables genome editing with reduced off-target activity
Yin, H., Song, C.-Q., Suresh, S., Kwan, S.-Y., Wu, Q., Walsh, S., Ding, J., Bogorad, R. L., Zhu, L. J., Wolfe, S. A., et al. (2018) · Nature Chemical Biology · doi:10.1038/nchembio.2559 · Cited by 240 · see citing works
A New Class of Medicines through DNA Editing
Porteus, M. H. (2019) · New England Journal of Medicine · doi:10.1056/nejmra1800729 · Cited by 229 · see citing works
Structural basis for Cas9 off-target activity
Pacesa, M., Lin, C.-H., Cléry, A., Saha, A., Arantes, P. R., Bargsten, K., Irby, M. J., Allain, F. H.-T., Palermo, G., Cameron, P., et al. (2022) · Cell · doi:10.1016/j.cell.2022.09.026 · Cited by 198 · see citing works
Prediction of the sequence-specific cleavage activity of Cas9 variants
Kim, N., Kim, H. K., Lee, S., Seo, J. H., Choi, J. W., Park, J., Min, S., Yoon, S., Cho, S.-R., & Kim, H. H. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0537-9 · Cited by 198 · see citing works
Highly Parallel Profiling of Cas9 Variant Specificity
Schmid-Burgk, J. L., Gao, L., Li, D., Gardner, Z., Strecker, J., Lash, B., & Zhang, F. (2020) · Molecular Cell · doi:10.1016/j.molcel.2020.02.023 · Cited by 197 · see citing works
Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity
Cromwell, C. R., Sung, K., Park, J., Krysler, A. R., Jovel, J., Kim, S. K., & Hubbard, B. P. (2018) · Nature Communications · doi:10.1038/s41467-018-03927-0 · Cited by 190 · see citing works
Off-target predictions in CRISPR-Cas9 gene editing using deep learning
Lin, J., & Wong, K.-C. (2018) · Bioinformatics · doi:10.1093/bioinformatics/bty554 · Cited by 185 · see citing works
Evaluating and enhancing target specificity of gene-editing nucleases and deaminases
Kim, D., Luk, K., Wolfe, S. A., & Kim, J.-S. (2019) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-013118-111730 · Cited by 171 · see citing works
Precision genome editing using cytosine and adenine base editors in mammalian cells
Huang, T. P., Newby, G. A., & Liu, D. R. (2021) · Nature Protocols · doi:10.1038/s41596-020-00450-9 · Cited by 175 · see citing works
Lau, C.-H., & Suh, Y. (2017) · F1000Research · doi:10.12688/f1000research.11243.1 · Cited by 161 · see citing works
Modrzejewski, D., Hartung, F., Lehnert, H., Sprink, T., Kohl, C., Keilwagen, J., & Wilhelm, R. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.574959 · Cited by 151 · see citing works
Kim, H. K., Lee, S., Kim, Y., Park, J., Min, S., Choi, J. W., Huang, T. P., Yoon, S., Liu, D. R., & Kim, H. H. (2020) · Nature Biomedical Engineering · doi:10.1038/s41551-019-0505-1 · Cited by 145 · see citing works
Palermo, G., Chen, J. S., Ricci, C. G., Rivalta, I., Jinek, M., Batista, V. S., Doudna, J. A., & McCammon, J. A. (2018) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583518000070 · Cited by 123 · see citing works
Self-citationExtension of the crRNA enhances Cpf1 gene editing in vitro and in vivo
Park, H. M., Liu, H., Wu, J., Chong, A., Mackley, V., Fellmann, C., Rao, A., Jiang, F., Chu, H., Murthy, N., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-05641-3 · Cited by 102 · see citing works
Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences
Ruffolo, J. A., Nayfach, S., Gallagher, J., Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E., Pacesa, M., et al. (2024) · doi:10.1101/2024.04.22.590591 · Cited by 90 · see citing works
Gene Therapy Intervention in Neovascular Eye Disease: A Recent Update
Lin, F.-L., Wang, P.-Y., Chuang, Y.-F., Wang, J.-H., Wong, V. H. Y., Bui, B. V., & Liu, G.-S. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.06.029 · Cited by 74 · see citing works
Donohoue, P. D., Pacesa, M., Lau, E., Vidal, B., Irby, M. J., Nyer, D. B., Rotstein, T., Banh, L., Toh, M. S., Gibson, J., et al. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.07.035 · Cited by 69 · see citing works
Genome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies
McTague, A., Rossignoli, G., Ferrini, A., Barral, S., & Kurian, M. A. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.630600 · Cited by 67 · see citing works
Genome-wide detection of CRISPR editing in vivo using GUIDE-tag
Liang, S.-Q., Liu, P., Smith, J. L., Mintzer, E., Maitland, S., Dong, X., Yang, Q., Lee, J., Haynes, C. M., Zhu, L. J., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-28135-9 · Cited by 54 · see citing works
PAM-flexible genome editing with an engineered chimeric Cas9
Zhao, L., Koseki, S. R. T., Silverstein, R. A., Amrani, N., Peng, C., Kramme, C., Savic, N., Pacesa, M., Rodríguez, T. C., Stan, T., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-41829-y · Cited by 59 · see citing works
Self-citationCurrent Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches
Leal, A., Herreno-Pachón, A., Benincore-Flórez, E., Karunathilaka, A., & Tomatsu, S. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25052456 · Cited by 58 · see citing works
Concerns regarding ‘off-target’activity of genome editing endonucleases
Kadam, U. S., Shelake, R. M., Chavhan, R. L., & Suprasanna, P. (2018) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2018.03.027 · Cited by 47 · see citing works
Kulcsár, P. I., Tálas, A., Tóth, E., Nyeste, A., Ligeti, Z., Welker, Z., & Welker, E. (2020) · Nature Communications · doi:10.1038/s41467-020-15021-5 · Cited by 45 · see citing works
crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays
Störtz, F., & Minary, P. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkaa885 · Cited by 37 · see citing works
Li, J., Wu, P., Cao, Z., Huang, G., Lu, Z., Yan, J., Zhang, H., Zhou, Y., Liu, R., Chen, H., et al. (2024) · Cell Reports · doi:10.1016/j.celrep.2024.113765 · Cited by 26 · see citing works
Okada, K., Aoki, K., Tabei, T., Sugio, K., Imai, K., Bonkohara, Y., & Kamachi, Y. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac100 · Cited by 14 · see citing works
Omura, S. N., Nakagawa, R., Kajimoto, S., Okazaki, S., Ishiguro, S., Mori, H., Onishi, K., Kashiwakura, Y., Hiramoto, T., Horinaka, K., et al. (2026) · Nature Communications · doi:10.1038/s41467-026-71626-2 · Cited by 2 · see citing works
Kinetic mechanism for fidelity of CRISPR-Cas9 variants
Hecht, A. D., & Igoshin, O. A. (2025) · bioRxiv · doi:10.1101/2025.01.27.634389
“A. Single-Molecule FRET Data and Processing Single-molecule FRET traces for wild-type, HF1, and eSpy(1.1)-Cas9 were obtained from the study of Chen et al. 2017 [1, 3].”
Influential
- Authors
- Chen, J. S., Dagdas, Y. S., Kleinstiver, B. P., Welch, M. M., Sousa, A. A., Harrington, L. B., Sternberg, S. H., Joung, J. K., Yildiz, A., & Doudna, J. A.
- Affiliations
- University of California, Berkeley (US)
- Harvard University (US)
- Massachusetts General Hospital (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A.
- ORCID
- Venue
- Nature
- Published
- 2017-09-20
- Volume
- 550
- Issue
- 7676
- Pages
- 407-410
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC5918688
- OpenAlex
- W2726053863
- Cited by
- 1,168
- References
- 29
- Citations by year
- 2026: 72 · 2025: 123 · 2024: 100 · 2023: 93 · 2022: 156 · 2021: 154 · 2020: 174 · 2019: 141 · 2018: 130 · 2017: 23
- FWCI
- 52.58
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- RePEc: Research Papers in Economics (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Science Foundation (1244557, 1617028)
- Howard Hughes Medical Institute
- Charles A. King Trust
- National Institutes of Health
- Natural Sciences and Engineering Research Council of Canada
- National Human Genome Research Institute (RM1HG009490)
- National Cancer Institute (K99CA218870)
- National Institute of General Medical Sciences (R01GM118773, R35GM118158, R01GM094522)
- 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 › DNA and Nucleic Acid Chemistry
- Keywords
- CRISPR/Cas9, SpCas9, off-target editing, genome editing specificity, HNH nuclease, nuclease activation, Streptococcus pyogenes
- MeSH terms
- Protein Domains, Gene Editing, Biotechnology, Endonucleases, Enzyme Activation, Humans, Streptococcus pyogenes, Substrate Specificity, Genetic Variation, Mutagenesis, CRISPR-Cas Systems, CRISPR-Associated Proteins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
