The DOI matches its Crossref record; 36 of the 36 references it cites were found in a registry or an index.
Engineered CRISPR-Cas9 nucleases with altered PAM specificities
Open accessAbstract
Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.
Overview
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
Verification
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?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Precision editing of alleles in a human cell has drastically improved the understanding of genotypic and phenotypic effects in gene therapy due to the lower off-target rates [119,120,121].”
From Utility of iPSC-Derived Cells for Disease Modeling, Drug Development, and Cell Therapy (2022) · cites this paper as [120]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Besides, there are other variants that could identify different PAM sequences, such as VQR, VRER, VRQR, and QQR1, allowing gene editing on a wider range of genetic loci.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [25]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“The basic bacterial selection scheme performed was adapted from a previously described positive selection system for identifying Cas9 mutants in E. coli31.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [31]
The cited source says what the claim attributes to it.
In the citing paper
“To be applicable to a wider range of genomic sites, some non-NGG PAM Cas9 were generated.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [25]
The cited source says what the claim attributes to it.
In the citing paper
“This could result in the development of wider-ranged VQR, EQR, and VRER SpCas9 variants, enabling the targeting of NGAN, NGNG, and NGCG PAMs, respectively (Kleinstiver et al., 2015; Hirano et al., 2016).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kleinstiver et al., 2015
The cited source says what the claim attributes to it.
In the citing paper
“These modifications have led to the creation of SpCas9 variants such as VQR, EQR, and VRER, which can recognize broader PAM sequences like NGAN, NGNG, and NGCG, respectively (Kleinstiver et al., 2015; Hirano et al., 2016).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kleinstiver et al., 2015
The cited source says what the claim attributes to it.
In the citing paper
“Cas9 genomic localization is delimited by the protospacer adjacent motif (PAM), which is NGG for SpCas9 while evolved variants of SpCas9 tolerate a broader range of PAM sequences17–21.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [18]
The check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“This screen identified four key beneficial point mutations in SpCas9, giving rise to evoCas9, a variant that exhibits fidelity exceeding SpCas9-HF1 and eSpCas9 (Kleinstiver et al., 2015; Casini et al., 2018).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Kleinstiver et al., 2015
Experimental — accuracy not yet measuredThe check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“SaKKHcas9 is an evolved variant of SaCas9 and recognizes the more relaxed NNNRRT PAM18.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [18]
Experimental — accuracy not yet measured
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
Fast and accurate short read alignment with Burrows-Wheeler transform
Li, H., & Durbin, R. (2009) · Bioinformatics · doi:10.1093/bioinformatics/btp324 · Cited by 58,607
Initial sequencing and analysis of the human genome
International Human Genome Sequencing Consortium, Whitehead Institute for Biomedical Research, C. F. G. R., Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., Devon, K., Dewar, K., et al. (2001) · Nature · doi:10.1038/35057062 · Cited by 24,216
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
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
RNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210
The new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013) · Cell · doi:10.1016/j.cell.2013.08.021 · Cited by 3,299
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
Self-citationCRISPR-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
Self-citationEfficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937
Self-citationIn vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687
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
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
Self-citationImproving 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
Self-citationDNA interrogation by the CRISPR RNA-guided endonuclease Cas9
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014) · Nature · doi:10.1038/nature13011 · Cited by 1,822
Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752
Short motif sequences determine the targets of the prokaryotic CRISPR defence system
Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J., & Almendros, C. (2009) · Microbiology · doi:10.1099/mic.0.023960-0 · Cited by 1,426
Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease
Anders, C., Niewoehner, O., Duerst, A., & Jinek, M. (2014) · Nature · doi:10.1038/nature13579 · Cited by 1,323
Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus
Deveau, H., Barrangou, R., Garneau, J. E., Labonté, J., Fremaux, C., Boyaval, P., Romero, D. A., Horvath, P., & Moineau, S. (2007) · Journal of Bacteriology · doi:10.1128/jb.01412-07 · Cited by 1,196
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
Self-citationDimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
Tsai, S. Q., Wyvekens, N., Khayter, C., Foden, J. A., Thapar, V., Reyon, D., Goodwin, M. J., Aryee, M. J., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2908 · Cited by 952
Self-citationGagnon, J. A., Valen, E., Thyme, S. B., Huang, P., Ahkmetova, L., Pauli, A., Montague, T. G., Zimmerman, S., Richter, C., & Schier, A. F. (2014) · PLoS ONE · doi:10.1371/journal.pone.0098186 · Cited by 893
Guilinger, J. P., Thompson, D. B., & Liu, D. R. (2014) · Nature Biotechnology · doi:10.1038/nbt.2909 · Cited by 836
Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J., & Church, G. M. (2013) · Nature Methods · doi:10.1038/nmeth.2681 · Cited by 786
Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus
Horvath, P., Romero, D. A., Coûté-Monvoisin, A.-C., Richards, M., Deveau, H., Moineau, S., Boyaval, P., Fremaux, C., & Barrangou, R. (2007) · Journal of Bacteriology · doi:10.1128/jb.01415-07 · Cited by 775
Optimization of scarless human stem cell genome editing
Yang, L., Güell, M., Byrne, S., Yang, J. L., Angeles, A. D. L., Mali, P., Aach, J., Kim-Kiselak, C., Briggs, A. W., Rios, X., et al. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt555 · Cited by 384
Saturation editing of genomic regions by multiplex homology-directed repair
Findlay, G. M., Boyle, E. A., Hause, R. J., Klein, J. C., & Shendure, J. (2014) · Nature · doi:10.1038/nature13695 · Cited by 362
The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
Chylinski, K., Le Rhun, A., & Charpentier, E. (2013) · RNA Biology · doi:10.4161/rna.24321 · Cited by 353
Protospacer recognition motifs: Mixed identities and functional diversity
Shah, S. A., Erdmann, S., Mojica, F. J. M., & Garrett, R. A. (2013) · RNA Biology · doi:10.4161/rna.23764 · Cited by 341
Fonfara, I., Le Rhun, A., Chylinski, K., Makarova, K. S., Lécrivain, A.-L., Bzdrenga, J., Koonin, E. V., & Charpentier, E. (2014) · Nucleic Acids Research · doi:10.1093/nar/gkt1074 · Cited by 328
Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells
Elliott, B., Richardson, C. A., Winderbaum, J., Nickoloff, J. A., & Jasin, M. (1998) · Molecular and Cellular Biology · doi:10.1128/mcb.18.1.93 · Cited by 305
A highly sensitive selection method for directed evolution of homing endonucleases
Chen, Z. (2005) · Nucleic Acids Research · doi:10.1093/nar/gni148 · Cited by 269
Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells
Zhang, Y., Ge, X., Yang, F., Zhang, L., Zheng, J., Tan, X., Jin, Z., Qu, J., & Gu, F. (2014) · Scientific Reports · doi:10.1038/srep05405 · Cited by 205
Directed Evolution and Substrate Specificity Profile of Homing Endonuclease I-SceI
Doyon, J. B., Pattanayak, V., Meyer, C. B., & Liu, D. R. (2006) · Journal of the American Chemical Society · doi:10.1021/ja057519l · Cited by 116
Kleinstiver, B. P., Fernandes, A. D., Gloor, G. B., & Edgell, D. R. (2010) · Nucleic Acids Research · doi:10.1093/nar/gkp1223 · Cited by 21
Self-citation
Cited by
Sorted by citations
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
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
Self-citationGenome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
Anzalone, A. V., Koblan, L. W., & Liu, D. R. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0561-9 · Cited by 2,237
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
Base editing: precision chemistry on the genome and transcriptome of living cells
Rees, H. A., & Liu, D. R. (2018) · Nature Reviews Genetics · doi:10.1038/s41576-018-0059-1 · Cited by 1,675
Metadata
- Authors
- 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., Aryee, M. J., & Joung, J. K.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Broad Institute (US)
- Corresponding author
- Joung, J. Keith
- ORCID
- Venue
- Nature
- Published
- 2015-06-22
- Volume
- 523
- Issue
- 7561
- Pages
- 481-485
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4540238
- OpenAlex
- W4245053291
- Semantic Scholar
- afe29e1767b5d0f3563ee9f2c1a586a9d5e8cb9b
- Cited by
- 1,624
- References
- 36
- Citations by year
- 2026: 58 · 2025: 105 · 2024: 117 · 2023: 134 · 2022: 166 · 2021: 165 · 2020: 164 · 2019: 154 · 2018: 147 · 2017: 186 · 2016: 167 · 2015: 58
- FWCI
- 80.95
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Institute of General Medical Sciences (DP1GM105378, R01GM107427, R01GM088040)
- National Center for Chronic Disease Prevention and Health Promotion (DP1 GM105378)
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, SpCas9, protospacer adjacent motif, genome editing, off-target effects, SaCas9, Cas9 nickase, zebrafish
- MeSH terms
- Animals, Cell Line, Humans, Mutation, Staphylococcus aureus, Streptococcus pyogenes, Substrate Specificity, Zebrafish, Protein Engineering, Genome, Directed Molecular Evolution, Amino Acid Substitution, Streptococcus thermophilus, Nucleotide Motifs, Clustered Regularly Interspaced Short Palindromic Repeats, 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
