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.
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Precision editing of alleles in a human cell has drastically improved the understanding of genotypic and phenotypic effects in gene therapy due to lower off-target rates.
“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]
The Abstract (page not provided) reports engineered Cas9 variants that edit human-cell gene sites and a variant with improved discrimination against off-target sites. It discusses allele-specific editing as a potential application, but does not report that allele editing improved understanding of genotypic or phenotypic effects in gene therapy, or establish that lower off-target rates caused such an improvement. (LLM, conf=0.86)
Experimental — accuracy not yet measuredOther variants such as VQR, VRER, VRQR, and QQR1 could identify different PAM sequences, allowing gene editing on a wider range of genetic loci.
“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]
The passage indexed as Abstract (page not provided) reports that VQR and VRER recognize different PAMs and enable editing at previously inaccessible sites. Our copy does not identify VRQR or QQR1 or establish that those two variants expand the targeting range. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe E. coli positive selection scheme used here was taken from an earlier system for finding Cas9 mutants.
“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]
Some non-NGG PAM Cas9 were generated to be applicable to a wider range of genomic sites.
“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]
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.
“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
These modifications have led to the creation of SpCas9 variants VQR, EQR, and VRER, which can recognize broader PAM sequences like NGAN, NGNG, and NGCG, respectively.
“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
Cas9 genomic localization is delimited by the PAM, which is NGG for SpCas9 while evolved variants of SpCas9 tolerate a broader range of PAM sequences.
“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]
evoCas9 exhibits fidelity exceeding SpCas9-HF1 and eSpCas9.
“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
The paper’s main text (page unavailable) describes a screen that produced VQR and EQR variants, and its specificity results compare D1135E with wild-type SpCas9. The passages searched for evoCas9, SpCas9-HF1, and eSpCas9 do not address whether evoCas9 has greater fidelity than those variants. (LLM, conf=0.76)
Experimental — accuracy not yet measuredSaKKHcas9 is an evolved variant of SaCas9 and recognizes the more relaxed NNNRRT PAM.
“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]
The Abstract reports PAMs for SaCas9 and suggests that its specificity could potentially be modified by mutagenesis and selection. A search of our copy for SaKKH, KKH and NNNRRT found no passage describing that variant or its PAM, so the attributed claim cannot be verified from this paper. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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“This screen identified four key beneficial point mutations in SpCas9, giving rise to evoCas9, a variant that exhibits fidelity exceeding SpCas9HF1 and eSpCas9 (Kleinstiver et al., 2015; Casini et al., 2018).”
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- 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 · 2014: 1 · 2013: 1
- 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
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