Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity
Open accessAbstract
Abstract Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2′,4′-BNA NC [N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNA NC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA–DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, “zipped” conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.
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?
Incorporation of bridged nucleic acids (2',4'-BNANC [N-Me]) or locked nucleic acids (LNA) into sgRNAs reduces the kinetics of Cas9 nuclease reactivity, thereby improving specificity of genome editing.
“Incorporation of bridged nucleic acids (2’,4’-BNANC [N-Me]) or locked nucleic acids (LNA) into the sgRNAs also reduces the kinetics of Cas9 nuclease reactivity, thereby improving the specificity of genome editing”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cromwell et al., 2018
The Abstract reports that BNANC-modified crRNAs slow Cas9 kinetics and improve specificity, and the Discussion links delayed cleavage to BNANC modification. The Results section on LNAs also reports improved specificity, but says Cas9 enzyme kinetics were reasonably similar with LNA-modified and unmodified crRNAs. The claimed kinetic reduction therefore does not hold for both modifications. (LLM, conf=0.89)
Experimental — accuracy not yet measuredChemical modifications on sgRNAs can influence their off-target effect.
“Apart from sgRNA length adjustment, chemical modifications on sgRNAs can also influence their off-target effect.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cromwell et al., 2018
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
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016) · Nature · doi:10.1038/nature17946 · Cited by 5,437 · see citing works
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153 · see citing works
Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1208507109 · Cited by 2,769 · see citing works
High-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
CRISPR/Cas, the Immune System of Bacteria and Archaea
Horvath, P., & Barrangou, R. (2010) · Science · doi:10.1126/science.1179555 · Cited by 2,471 · see citing works
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2508 · Cited by 2,429 · see citing works
Rationally 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
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
CRISPR–Cas9 Structures and Mechanisms
Jiang, F., & Doudna, J. A. (2017) · Annual Review of Biophysics · doi:10.1146/annurev-biophys-062215-010822 · Cited by 2,029 · see citing works
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2808 · Cited by 1,993 · see citing works
Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R. (2013) · Nature Biotechnology · doi:10.1038/nbt.2673 · Cited by 1,487 · see citing works
Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., Maeder, M. L., Joung, J. K., Chen, Z.-Y., & Liu, D. R. (2015) · Nature Biotechnology · doi:10.1038/nbt.3081 · Cited by 1,460 · see citing works
Structural 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
Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
Joung, J., Konermann, S., Gootenberg, J. S., Abudayyeh, O. O., Platt, R. J., Brigham, M. D., Sanjana, N. E., & Zhang, F. (2017) · Nature Protocols · doi:10.1038/nprot.2017.016 · Cited by 1,243 · see citing works
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
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. (2017) · Nature · doi:10.1038/nature24268 · Cited by 1,168 · see citing works
Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells
Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3290 · Cited by 1,090 · see citing works
Guilinger, J. P., Thompson, D. B., & Liu, D. R. (2014) · Nature Biotechnology · doi:10.1038/nbt.2909 · Cited by 836 · see citing works
LNA (Locked Nucleic Acid): High-Affinity Targeting of Complementary RNA and DNA
Vester, B., & Wengel, J. (2004) · Biochemistry · doi:10.1021/bi0485732 · Cited by 727 · 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
Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality
Elmén, J. (2005) · Nucleic Acids Research · doi:10.1093/nar/gki193 · Cited by 563 · see citing works
A 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
A Rapid and General Assay for Monitoring Endogenous Gene Modification
Guschin, D. Y., Waite, A. J., Katibah, G. E., Miller, J. C., Holmes, M. C., & Rebar, E. J. (2010) · Methods in molecular biology · doi:10.1007/978-1-60761-753-2_15 · Cited by 500 · see citing works
Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing
Yin, H., Song, C.-Q., Suresh, S., Wu, Q., Walsh, S., Rhym, L. H., Mintzer, E., Bolukbasi, M. F., Zhu, L. J., Kauffman, K., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.4005 · Cited by 499 · see citing works
Swayze, E. E., Siwkowski, A. M., Wancewicz, E. V., Migawa, M. T., Wyrzykiewicz, T. K., Hung, G., Monia, B. P., & Bennett, A. C. F. (2006) · Nucleic Acids Research · doi:10.1093/nar/gkl1071 · Cited by 473 · see citing works
Klann, T. S., Black, J. B., Chellappan, M., Safi, A., Song, L., Hilton, I. B., Crawford, G. E., Reddy, T. E., & Gersbach, C. A. (2017) · Nature Biotechnology · doi:10.1038/nbt.3853 · Cited by 447 · see citing works
Wang, X., Wang, Y., Wu, X., Wang, J., Wang, Y., Qiu, Z., Chang, T., Huang, H., Lin, R.-J., & Yee, J.-K. (2015) · Nature Biotechnology · doi:10.1038/nbt.3127 · Cited by 442 · see citing works
Cas-analyzer: an online tool for assessing genome editing results using NGS data
Park, J., Lim, K., Kim, J., & Bae, S. (2016) · Bioinformatics · doi:10.1093/bioinformatics/btw561 · Cited by 400 · see citing works
Self-citationDesign of LNA probes that improve mismatch discrimination
You, Y. (2006) · Nucleic Acids Research · doi:10.1093/nar/gkl175 · Cited by 338 · see citing works
Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity
Tycko, J., Myer, V. E., & Hsu, P. D. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.07.004 · Cited by 304 · see citing works
CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells
Hoban, M. D., Lumaquin, D., Kuo, C. Y., Romero, Z., Long, J., Ho, M., Young, C. S., Mojadidi, M., Fitz-Gibbon, S., Cooper, A. R., et al. (2016) · Molecular Therapy · doi:10.1038/mt.2016.148 · Cited by 220 · see citing works
A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
O'Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., & Segal, D. J. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv137 · Cited by 219 · see citing works
Guilinger, J. P., Pattanayak, V., Reyon, D., Tsai, S. Q., Sander, J. D., Joung, J. K., & Liu, D. R. (2014) · Nature Methods · doi:10.1038/nmeth.2845 · Cited by 213 · see citing works
Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq
Kim, D., Kim, S., Kim, S., Park, J., & Kim, J.-S. (2016) · Genome Research · doi:10.1101/gr.199588.115 · Cited by 208 · see citing works
Self-citationSynthetic CRISPR RNA-Cas9–guided genome editing in human cells
Rahdar, M., McMahon, M. A., Prakash, T. P., Swayze, E. E., Bennett, C. F., & Cleveland, D. W. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1520883112 · Cited by 183 · see citing works
Design, Synthesis, and Properties of 2‘,4‘-BNA NC : A Bridged Nucleic Acid Analogue
Rahman, S. M. A., Seki, S., Obika, S., Yoshikawa, H., Miyashita, K., & Imanishi, T. (2008) · Journal of the American Chemical Society · doi:10.1021/ja710342q · Cited by 164 · see citing works
2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA)
Obika, S., Uneda, T., Sugimoto, T., Nanbu, D., Minami, T., Doi, T., & Imanishi, T. (2001) · Bioorganic & Medicinal Chemistry · doi:10.1016/s0968-0896(00)00325-4 · Cited by 151 · see citing works
Continuous directed evolution of DNA-binding proteins to improve TALEN specificity
Hubbard, B. P., Badran, A. H., Zuris, J. A., Guilinger, J. P., Davis, K. M., Chen, L., Tsai, S. Q., Sander, J. D., Joung, J. K., & Liu, D. R. (2015) · Nature Methods · doi:10.1038/nmeth.3515 · Cited by 121 · see citing works
Self-citationStructural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
Lim, Y., Bak, S. Y., Sung, K., Jeong, E., Lee, S. H., Kim, J., Bae, S., & Kim, S. K. (2016) · Nature Communications · doi:10.1038/ncomms13350 · Cited by 115 · see citing works
Self-citationHow specific is CRISPR/Cas9 really?
O’Geen, H., Yu, A. S., & Segal, D. J. (2015) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2015.10.001 · Cited by 108 · see citing works
Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules
Klein, M., Eslami-Mossallam, B., Arroyo, D. G., & Depken, M. (2018) · Cell Reports · doi:10.1016/j.celrep.2018.01.045 · Cited by 107 · see citing works
Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders
Christie, K. A., Courtney, D., DeDionisio, L., Chao-Shern, C., Majumdar, S. D., Mairs, L., Nesbit, M. A., & Moore, T. (2017) · Scientific Reports · doi:10.1038/s41598-017-16279-4 · Cited by 82 · see citing works
Xu, X., Duan, D., & Chen, S. (2017) · Scientific Reports · doi:10.1038/s41598-017-00180-1 · Cited by 78 · see citing works
Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system
Cheong, T.-C., Compagno, M., & Chiarle, R. (2016) · Nature Communications · doi:10.1038/ncomms10934 · Cited by 71 · see citing works
Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease
Rueda, F. O., Biśta, M., Newton, M. D., Goeppert, A. U., Cuomo, M. E., Gordon, E., Kroener, F., Read, J. A., Wrigley, J. D. J., Rueda, D., et al. (2017) · Nature Communications · doi:10.1038/s41467-017-01732-9 · Cited by 69 · see citing works
Gotfredsen, C. H., Schultze, P., & Feigon, J. F. (1998) · Journal of the American Chemical Society · doi:10.1021/ja973221m · Cited by 46 · see citing works
CRISPR-DAV: CRISPR NGS data analysis and visualization pipeline
Wang, X., Tilford, C. A., Neuhaus, I., Mintier, G. A., Guo, Q. J., Feder, J. N., & Kirov, S. A. (2017) · Bioinformatics · doi:10.1093/bioinformatics/btx518 · Cited by 45 · see citing works
Anders, C., Niewoehner, O., & Jínek, M. (2015) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/bs.mie.2015.02.008 · Cited by 30 · see citing works
BNANC Gapmers Revert Splicing and Reduce RNA Foci with Low Toxicity in Myotonic Dystrophy Cells
Manning, K. S., Rao, A. N., Castro, M., & Cooper, T. A. (2017) · ACS Chemical Biology · doi:10.1021/acschembio.7b00416 · Cited by 20 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
RNA-based therapeutics: an overview and prospectus
Zhu, Y., Zhu, L., Wang, X., & Jin, H. (2022) · Cell Death and Disease · doi:10.1038/s41419-022-05075-2 · Cited by 658 · 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
“[N-Me]) or locked nucleic acids (LNA) into the sgRNAs also reduces the kinetics of Cas9 nuclease reactivity, thereby improving the specificity of genome editing (Cromwell et al., 2018).”
BackgroundCRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors
Wang, D., Zhang, F., & Gao, G. (2020) · Cell · doi:10.1016/j.cell.2020.03.023 · Cited by 526 · see citing works
Latest 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
Recent progress in non-native nucleic acid modifications
McKenzie, L. K., El-Khoury, R., Thorpe, J. D., Damha, M. J., & Hollenstein, M. (2021) · Chemical Society Reviews · doi:10.1039/d0cs01430c · Cited by 300 · see citing works
Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Tao, J., Bauer, D. E., & Chiarle, R. (2023) · Nature Communications · doi:10.1038/s41467-023-35886-6 · Cited by 215 · 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
Development of CRISPR-Cas systems for genome editing and beyond
Zhang, F. (2019) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583519000052 · Cited by 185 · see citing works
Chemically Modified Platforms for Better RNA Therapeutics
Shi, Y., Zhen, X., Zhang, Y., Li, Y., Koo, S., Saiding, Q., Kong, N., Liu, G., Chen, W., & Tao, W. (2024) · Chemical Reviews · doi:10.1021/acs.chemrev.3c00611 · Cited by 184 · 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
CRISPR-Cas9-mediated homology-directed repair for precise gene editing
Liao, H., Wu, J., VanDusen, N. J., Li, Y., & Zheng, Y. (2024) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2024.102344 · Cited by 181 · see citing works
Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing
Han, H. A., Pang, J. K. S., & Soh, B.-S. (2020) · Journal of Molecular Medicine · doi:10.1007/s00109-020-01893-z · Cited by 143 · see citing works
CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences
Karlson, C. K. S., Noor, S. N. M., Nolte, N., & Tan, B. C. (2021) · Plants · doi:10.3390/plants10102055 · Cited by 126 · see citing works
CRISPR Modeling and Correction of Cardiovascular Disease
Liu, N., & Olson, E. N. (2022) · Circulation Research · doi:10.1161/circresaha.122.320496 · Cited by 118 · see citing works
Improving CRISPR Genome Editing by Engineering Guide RNAs
Moon, S. B., Kim, D. Y., Kim, J., Kim, J., & Kim, Y.-S. (2019) · Trends in biotechnology · doi:10.1016/j.tibtech.2019.01.009 · Cited by 104 · see citing works
Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies
Castro, N. G. G., Bjelic, J., Malhotra, G., Huang, C., & Alsaffar, S. H. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms221910355 · Cited by 107 · see citing works
Haghighi, E., Abolmaali, S. S., Dehshahri, A., Shaegh, S. A. M., Azarpira, N., & Tamaddon, A. M. (2024) · Journal of Nanobiotechnology · doi:10.1186/s12951-024-02972-w · Cited by 111 · see citing works
Kimberland, M. L., Hou, W., Alfonso-Pecchio, A. R., Wilson, S. P., Rao, Y., Zhang, S., & Lu, Q. (2018) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2018.08.007 · Cited by 96 · see citing works
Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems
Filippova, J. A., Matveeva, A. M., Zhuravlev, E. S., & Stepanov, G. A. (2019) · Biochimie · doi:10.1016/j.biochi.2019.09.003 · Cited by 95 · see citing works
Conditional control of RNA-guided nucleic acid cleavage and gene editing
Wang, S., Wu, L., Huang, H., Xiong, W., Liu, J., Wei, L., Yin, P., Tian, T., & Zhou, X. (2020) · Nature Communications · doi:10.1038/s41467-019-13765-3 · Cited by 96 · see citing works
Chen, Q., Zhang, Y., & Yin, H. (2020) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2020.10.014 · Cited by 97 · see citing works
Chemical Synthesis and Biological Application of Modified Oligonucleotides
Glazier, D. A., Liao, J., Roberts, B. L., Li, X., Yang, K., Stevens, C. M., & Tang, W. (2020) · Bioconjugate Chemistry · doi:10.1021/acs.bioconjchem.0c00060 · Cited by 91 · see citing works
RNA chemistry and therapeutics
Wang, S., Weissman, D., & Dong, Y. (2025) · Nature Reviews Drug Discovery · doi:10.1038/s41573-025-01237-x · Cited by 98 · see citing works
CRISPR/Cas9 Landscape: Current State and Future Perspectives
Tyumentsev, A. I., Tyumentsev, A. I., & Акимкин, В. Г. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242216077 · Cited by 95 · see citing works
Chemical Modifications of CRISPR RNAs to Improve Gene-Editing Activity and Specificity
Rozners, E. (2022) · Journal of the American Chemical Society · doi:10.1021/jacs.2c02633 · Cited by 85 · see citing works
Allen, D. G., Rosenberg, M., & Hendel, A. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2020.617910 · Cited by 82 · see citing works
Lopes, R., & Prasad, M. K. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1339189 · Cited by 82 · see citing works
Ke, Y., Ghalandari, B., Huang, S., Li, S., Huang, C., Zhi, X., Cui, D., & Ding, X. (2022) · Chemical Science · doi:10.1039/d1sc06832f · Cited by 78 · see citing works
CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer
Azangou‐Khyavy, M., Ghasemi, M., Khanali, J., Boroomand-Saboor, M., Jamalkhah, M., Soleimani, M., & Kiani, J. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.02062 · Cited by 77 · see citing works
Precise genome-editing in human diseases: mechanisms, strategies and applications
Zheng, Y., Li, Y., Zhou, K., Li, T., VanDusen, N. J., & Hua, Y. (2024) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-024-01750-2 · Cited by 80 · see citing works
Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine
Azeez, S. S., Hamad, R. S., Hamad, B., Shekha, M. S., & Bergsten, P. (2024) · Frontiers in Genome Editing · doi:10.3389/fgeed.2024.1509924 · Cited by 84 · 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
Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells
Méndez-Mancilla, A., Wessels, H.-H., Legut, M., Kadina, A., Mabuchi, M., Walker, J., Robb, G. B., Holden, K., & Sanjana, N. E. (2022) · Cell Chemical Biology · doi:10.1016/j.chembiol.2021.07.011 · Cited by 68 · see citing works
Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA
Zhang, Y., Ling, X., Su, X., Zhang, S., 王, J. 璟. W., Zhang, P., Feng, W., Zhu, Y. Y., Liu, T., & Tang, X. (2020) · Angewandte Chemie International Edition · doi:10.1002/anie.202009890 · Cited by 67 · see citing works
CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges
Demirci, S., Leonard, A. K., Haro‐Mora, J. J., Uchida, N., & Tisdale, J. F. (2019) · Advances in experimental medicine and biology · doi:10.1007/5584_2018_331 · Cited by 67 · see citing works
Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering
Huang, X., Yang, D., Zhang, J., Xu, J., & Chen, Y. E. (2022) · Cells · doi:10.3390/cells11142186 · Cited by 65 · see citing works
Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing
Li, J., Hong, S., Chen, W., Zuo, E., & Yang, H. (2019) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2019.11.002 · Cited by 63 · see citing works
Taemaitree, L., Shivalingam, A., El-Sagheer, A. H., & Brown, T. P. (2019) · Nature Communications · doi:10.1038/s41467-019-09600-4 · Cited by 61 · see citing works
O’Reilly, D., Kartje, Z. J., Ageely, E. A., Malek-Adamian, E., Habibian, M., Schofield, A., Barkau, C. L., Rohilla, K. J., DeRossett, L. B., Weigle, A. T., et al. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky1214 · Cited by 61 · see citing works
Cas9 deactivation with photocleavable guide RNAs
Zou, R. S., Liu, Y., Wu, B., & Ha, T. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.02.007 · Cited by 60 · see citing works
Molecular mechanisms, off‐target activities, and clinical potentials of genome editing systems
Zheng, N., Li, L., & Wang, X. (2020) · Clinical and Translational Medicine · doi:10.1002/ctm2.34 · Cited by 56 · see citing works
Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing
Liu, R., Liang, L., Freed, E. F., & Gill, R. T. (2020) · Trends in biotechnology · doi:10.1016/j.tibtech.2020.07.005 · Cited by 56 · see citing works
Sun, Y., Chen, W., Liu, J., Li, J., Zhang, Y., Cai, W., Liu, L., Tang, X., Hou, J., Wang, M., et al. (2022) · Angewandte Chemie International Edition · doi:10.1002/anie.202212413 · Cited by 55 · see citing works
Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA
Hu, Z., Sun, A., Yang, J., Naz, G., Sun, G., Li, Z., Liu, J., Zhang, S., & Zhang, X. (2023) · Chemical Science · doi:10.1039/d3sc00629h · Cited by 55 · see citing works
Chemical engineering of CRISPR–Cas systems for therapeutic application
Barber, H. M., Pater, A. A., Gagnon, K. T., Damha, M. J., & O’Reilly, D. (2024) · Nature Reviews Drug Discovery · doi:10.1038/s41573-024-01086-0 · Cited by 55 · see citing works
Bollen, Y., Post, J. B., Koo, B., & Snippert, H. J. G. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky571 · Cited by 52 · see citing works
Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences
Krysler, A. R., Cromwell, C. R., Tu, T., Jovel, J., & Hubbard, B. P. (2022) · Nature Communications · doi:10.1038/s41467-022-29202-x · Cited by 48 · see citing works
Self-citationSpecificity of oligonucleotide gene therapy (OGT) agents
Nedorezova, D. D., Dubovichenko, M. V., Belyaeva, E. P., Grigorieva, E. D., Peresadina, A. V., & Kolpashchikov, D. M. (2022) · Theranostics · doi:10.7150/thno.77830 · Cited by 48 · see citing works
CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering
Lone, B. A., Karna, S. K. L., Ahmad, F., Shahi, N., & Pokharel, Y. R. (2018) · Genetics Research International · doi:10.1155/2018/3797214 · Cited by 48 · see citing works
Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage
Zuo, Z., & Liu, J. (2020) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2020.01.013 · Cited by 48 · see citing works
Okafor, I. C., Singh, D., Wang, Y., Jung, M., Wang, H., Mallon, J. P., Bailey, S., Lee, J. K., & Ha, T. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz1058 · Cited by 44 · see citing works
Natural, modified and conjugated carbohydrates in nucleic acids
Dhara, D., Mulard, L. A., & Hollenstein, M. (2025) · Chemical Society Reviews · doi:10.1039/d4cs00799a · Cited by 44 · see citing works
Regulating CRISPR/Cas9 Function through Conditional Guide RNA Control
Brown, W., Zhou, W., & Deiters, A. (2020) · ChemBioChem · doi:10.1002/cbic.202000423 · Cited by 41 · see citing works
Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain
Sung, K., Park, J., Kim, Y.-G., Lee, N. K., & Kim, S. K. (2018) · Journal of the American Chemical Society · doi:10.1021/jacs.8b03102 · Cited by 41 · see citing works
Self-citationCRISPR RNA-guided autonomous delivery of Cas9
Wilkinson, R. A., Martin, C. D., Nemudryi, A. A., & Wiedenheft, B. (2018) · Nature Structural & Molecular Biology · doi:10.1038/s41594-018-0173-y · Cited by 42 · see citing works
Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles
Ageely, E. A., Chilamkurthy, R., Jana, S. K., Abdullahu, L., O’Reilly, D., Jensik, P. J., Damha, M. J., & Gagnon, K. T. (2021) · Nature Communications · doi:10.1038/s41467-021-26989-z · Cited by 39 · see citing works
Paving the way towards precise and safe CRISPR genome editing
Śledziński, P., Dabrowska, M., Nowaczyk, M., & Olejniczak, M. (2021) · Biotechnology Advances · doi:10.1016/j.biotechadv.2021.107737 · Cited by 41 · see citing works
CRISPR/Cas9 as a therapeutic tool for triple negative breast cancer: from bench to clinics
Tiwari, P. K., Ko, T.-H., Dubey, R., Chouhan, M., Tsai, L.-W., Singh, H. N., Chaubey, K. K., Dayal, D., Chiang, C., & Kumar, S. (2023) · Frontiers in Molecular Biosciences · doi:10.3389/fmolb.2023.1214489 · Cited by 41 · see citing works
Xiao, H., Hu, J., Huang, C., Feng, W., Liu, Y., Kumblathan, T., Tao, J., Xu, J., Le, X. C., & Zhang, H. (2023) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2023.117000 · Cited by 38 · see citing works
CRISPR/Cas9 gene editing therapies for cystic fibrosis
Graham, C., & Hart, S. L. (2021) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2021.1869208 · Cited by 38 · see citing works
Exploring Parkinson-associated kinases for CRISPR/Cas9-based gene editing: beyond alpha-synuclein
Mansour, H. M., & El-Khatib, A. S. (2023) · Ageing Research Reviews · doi:10.1016/j.arr.2023.102114 · Cited by 36 · see citing works
Matrix metalloproteinase‐2 mediates ribosomal RNA transcription by cleaving nucleolar histones
Ali, M. A., Garcia‐Vilas, J. A., Cromwell, C. R., Hubbard, B. P., Hendzel, M. J., & Schulz, R. (2021) · FEBS Journal · doi:10.1111/febs.16061 · Cited by 36 · see citing works
Self-citationMerlin, J. P. J., & Abrahamse, H. (2024) · Biomedicine & Pharmacotherapy · doi:10.1016/j.biopha.2024.117516 · Cited by 35 · see citing works
Recent advances in CRISPR-based single-nucleotide fidelity diagnostics
Kohabir, K. A. V., Sistermans, E. A., & Wolthuis, R. M. F. (2025) · Communications Medicine · doi:10.1038/s43856-025-00933-4 · Cited by 39 · see citing works
Emerging Gene Therapeutics for Epidermolysis Bullosa under Development
Bischof, J., Hierl, M., & Koller, U. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25042243 · Cited by 35 · see citing works
Mao, X., Xu, J., Jiang, J., Li, Q., Yao, P., Jiang, J., Gong, L., Dong, Y., Tu, B., Wang, R., et al. (2024) · Communications Biology · doi:10.1038/s42003-024-07173-7 · Cited by 33 · see citing works
van de Vrugt, H. J., Harmsen, T., Riepsaame, J., Alexantya, G., van Mil, S. E., de Vries, Y., Ali, R. B., Huijbers, I. J., Dorsman, J. C., Wolthuis, R. M. F., et al. (2019) · Scientific Reports · doi:10.1038/s41598-018-36506-w · Cited by 31 · see citing works
Ma, Z., Ma, L., & Zhou, J. (2023) · Molecular Horticulture · doi:10.1186/s43897-023-00049-0 · Cited by 36 · see citing works
Development of aptamer-based inhibitors for CRISPR/Cas system
Zhao, J. C., Inomata, R., Kato, Y., & Miyagishi, M. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa865 · Cited by 31 · see citing works
Current advances in overcoming obstacles of CRISPR/Cas9 off-target genome editing
Aquino‐Jarquín, G. (2021) · Molecular Genetics and Metabolism · doi:10.1016/j.ymgme.2021.08.002 · Cited by 32 · see citing works
Bridged Nucleic Acids Reloaded
Soler‐Bistué, A., Zorreguieta, Á., & Tolmasky, M. E. (2019) · Molecules · doi:10.3390/molecules24122297 · Cited by 31 · see citing works
Harnessing noncanonical crRNA for highly efficient genome editing
Xun, G., Zhu, Z., Singh, N., Lu, J., Jain, P., & Zhao, H. (2024) · Nature Communications · doi:10.1038/s41467-024-48012-x · Cited by 30 · see citing works
Controlling CRISPR‐Cas9 by guide RNA engineering
Hu, L., Li, Y., Wang, J., Zhao, Y., & Wang, Y. (2022) · Wiley Interdisciplinary Reviews - RNA · doi:10.1002/wrna.1731 · Cited by 29 · see citing works
Sakovina, L., Vokhtantsev, I. P., Vorobyeva, M., Vorobjev, P., & Новопашина, Д. С. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms232113460 · Cited by 29 · see citing works
Engineering guide RNA to reduce the off-target effects of CRISPR
Wu, J., & Yin, H. (2019) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2019.11.003 · Cited by 27 · see citing works
Li, Y., Hu, Q., Bai, M., Qing, M., & Bai, L. (2025) · Analytical Chemistry · doi:10.1021/acs.analchem.4c06107 · Cited by 26 · see citing works
Fu, S., Li, J., Chen, J., Zhang, L., Liu, J., Liu, H., & Su, X. (2024) · Nature Biotechnology · doi:10.1038/s41587-024-02388-9 · Cited by 25 · see citing works
CRISPR/Cas technologies for cancer drug discovery and treatment
Wang, K. C., Zheng, T., & Hubbard, B. P. (2025) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2025.02.009 · Cited by 25 · see citing works
Self-citationAllen, D. G., Weiss, L. E., Saguy, A., Rosenberg, M., Iancu, O., Matalon, O., Lee, C. M., Beider, K., Nagler, A., Shechtman, Y., et al. (2022) · The CRISPR Journal · doi:10.1089/crispr.2021.0128 · Cited by 24 · see citing works
Lipid phosphate phosphatase-2 promotes tumor growth through increased c-Myc expression
Tang, X., Cromwell, C. R., Liu, R., Godbout, R., Hubbard, B. P., McMullen, T. P. W., & Brindley, D. N. (2022) · Theranostics · doi:10.7150/thno.66230 · Cited by 23 · see citing works
Self-citationThe advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research
Zhang, J., Khazalwa, E. M., Abkallo, H. M., Zhou, Y., Nie, X., Ruan, J., Zhao, C., Wang, J., Xu, J., Li, X., et al. (2021) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2021.03.015 · Cited by 25 · see citing works
From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods
Bazaz, M. R., & Dehghani, H. (2022) · Life Sciences · doi:10.1016/j.lfs.2022.120409 · Cited by 22 · see citing works
Acceleration of cancer science with genome editing and related technologies
Sakuma, T., & Yamamoto, T. (2018) · Cancer Science · doi:10.1111/cas.13832 · Cited by 23 · see citing works
CRISPR‐Cas9: A cornerstone for the evolution of precision medicine
Razzouk, S. (2018) · Annals of Human Genetics · doi:10.1111/ahg.12271 · Cited by 22 · see citing works
Ha, D.-I., Lee, J. M., Lee, N., Kim, D., Ko, J., & Kim, Y.-S. (2020) · Experimental & Molecular Medicine · doi:10.1038/s12276-020-00521-7 · Cited by 21 · see citing works
Review, analysis, and optimization of the CRISPR Streptococcus pyogenes Cas9 system
Wang, G., & Li, J. (2021) · Medicine in Drug Discovery · doi:10.1016/j.medidd.2021.100080 · Cited by 22 · see citing works
Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing
Cai, W., & Wang, M. (2019) · 中国科学通报:英文版 · doi:10.1016/j.scib.2019.07.035 · Cited by 21 · see citing works
Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration
Zhang, M., Yang, C., Tasan, I., & Zhao, H. (2021) · ACS Synthetic Biology · doi:10.1021/acssynbio.0c00576 · Cited by 20 · see citing works
Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA
Zhang, Y., Ling, X., Su, X., Zhang, S., Wang, J., Zhang, P., Feng, W., Zhu, Y. Y., Liu, T., & Tang, X. (2020) · Angewandte Chemie · doi:10.1002/ange.202009890 · Cited by 21 · see citing works
Unlocking the Potential of Chemically Modified Nucleic Acid Therapeutics
Gao, J., Nutan, B., Gargouri, D., Pisal, N. D., Do, V., Zubair, M., Alanzi, H., Wang, H., Lee, D., Joshi, N., et al. (2024) · Advanced Therapeutics · doi:10.1002/adtp.202400231 · Cited by 20 · see citing works
Therapeutic gene editing strategies using CRISPR-Cas9 for the β-hemoglobinopathies
Papizan, J. B., Porter, S. N., Sharma, A., & Pruett‐Miller, S. M. (2020) · Journal of Biomedical Research · doi:10.7555/jbr.34.20200096 · Cited by 19 · see citing works
Tripeptide IRW Upregulates NAMPT Protein Levels in Cells and Obese C57BL/6J Mice
Bhullar, K. S., Son, M., Kerek, E. M., Cromwell, C. R., Wingert, B. M., Wu, K., Jovel, J., Camacho, C. J., Hubbard, B. P., & Wu, J. (2021) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.0c07831 · Cited by 19 · see citing works
Self-citationThermophilic Nucleic Acid Polymerases and Their Application in Xenobiology
Wang, G., Du, Y., Ma, X., Ye, F., Qin, Y., Wang, Y., Xiang, Y., Tao, R. X., & Chen, T. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms232314969 · Cited by 19 · see citing works
Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo
Zhang, H., Kelly, K. L., Lee, J. L. C., Echeverria, D., Cooper, D., Panwala, R., Amrani, N., Chen, Z., Gaston, N., Wagh, A. A., et al. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad1125 · Cited by 19 · see citing works
Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA
Qi, Q., Liu, X., Xiong, W., Zhang, K., Shen, W., Zhang, Y., Xu, X., Zhong, C., Zhang, Y., Tian, T., et al. (2024) · Cell chemical biology · doi:10.1016/j.chembiol.2024.09.006 · Cited by 18 · see citing works
Introduction and History of the Chemistry of Nucleic Acids Therapeutics
Gait, M. J., & Agrawal, S. (2022) · Methods in molecular biology · doi:10.1007/978-1-0716-2010-6_1 · Cited by 17 · see citing works
Thorpe, C., Epple, S., Woods, B., El-Sagheer, A. H., & Brown, T. P. (2019) · Organic & Biomolecular Chemistry · doi:10.1039/c9ob00691e · Cited by 17 · see citing works
CRISPR, animals, and FDA oversight: Building a path to success
Epstein, L. R., Lee, S. S., Miller, M. F., & Lombardi, H. A. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2004831117 · Cited by 17 · see citing works
Bak, S. Y., Jung, Y., Park, J., Sung, K., Jang, H., Bae, S., & Kim, S. K. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab858 · Cited by 17 · see citing works
Self-citationViral and nonviral nanocarriers for in vivo CRISPR-based gene editing
Guo, Z., Zhu, A. T., Fang, R. H., & Zhang, L. (2024) · Nano Research · doi:10.1007/s12274-024-6748-5 · Cited by 17 · see citing works
- Authors
- Cromwell, C. R., Sung, K., Park, J., Krysler, A. R., Jovel, J., Kim, S. K., & Hubbard, B. P.
- Affiliations
- University of Alberta (CA)
- Seoul National University (KR)
- Corresponding author
- Hubbard, Basil P.
- ORCID
- Venue
- Nature Communications
- Published
- 2018-04-13
- Volume
- 9
- Issue
- 1
- Pages
- 1448-1448
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC5899152
- OpenAlex
- W2796961959
- Cited by
- 190
- References
- 50
- Citations by year
- 2026: 13 · 2025: 15 · 2024: 29 · 2023: 19 · 2022: 30 · 2021: 33 · 2020: 26 · 2019: 17 · 2018: 8
- FWCI
- 7.47
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Universities Space Research Association
- National Research Foundation
- National Research Foundation of Korea (2018R1A2B2001422)
- Natural Sciences and Engineering Research Council of Canada (rgpin-2016-06381, RGPIN-2016)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Keywords
- CRISPR/Cas9, bridged nucleic acids, locked nucleic acid, single-molecule FRET, dwell time, synthetic nucleic acids
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, DNA, Endonucleases, Escherichia coli, HeLa Cells, Humans, RNA, Sensitivity and Specificity, Streptococcus pyogenes, RNA, Guide, Kinetoplastida, Polymorphism, Single Nucleotide, Fluorescence Resonance Energy Transfer, Cell Line, Tumor, DNA Cleavage, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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