ArticleNature2015
Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
Article in Nature, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 936 papers, 1 of them a synthesis that pooled it.
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Who cites it
936 citing papers in PubMed, 1 synthesis or guideline pooled it.
- The evolving landscape of gene editing therapies for human genetic diseases: a twenty-year bibliometric analysis.Frontiers in medicine · 2026Pooled it
- Disruption ofVirulence · 2026Article
- Translating base editors: a decade of progress from the bench to the clinic.Nature reviews. Drug discovery · 2026Review
- Rational selection of SpCas9 and variant-derived adenine base editors for human genome editing.Gene therapy · 2026Article
- Structure-guided discovery and engineering of natural Cas9 orthologs for high-fidelity editing.Science China. Life sciences · 2026Article
- Base editing for precision therapeutics.Cell genomics · 2026Review
- SLC25A38 gene modification mediated by CRISPR/Cas9 in HEK293T cell line.Journal of applied genetics · 2026Article
- CRISPR/Cas system as a novel therapeutic strategy to combat multi-drug-resistant bacteria.Archives of microbiology · 2026Review
- Constructing an Adenine Base Editor inMicroorganisms · 2026Article
- A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos.Marine biotechnology (New York, N.Y.) · 2026Article
- A highly penetrantbioRxiv : the preprint server for biology · 2026Article
- In Situ Amplified Mutational mRNA Imaging Using a Spatially Confined CRISPR Nanoplatform.Angewandte Chemie (International ed. in English) · 2026Article
- The future of pediatric gene therapy: CRISPR-Cas9, AI, and personalized medicine.Pediatric research · 2026Review
- iSCORE-PD: an isogenic stem cell collection to research Parkinson's disease.Nature communications · 2026Article
- Engineered transformer base editor with unconstrained PAM requirements.Molecular therapy. Nucleic acids · 2026Article
- Machine learning-driven optimization of specific, compact, and efficient base editors via single-round diversification.Nucleic acids research · 2026Article
- A primer on prime: A prime editing update from advances to first-in-human trial.Molecular therapy : the journal of the American Society of Gene Therapy · 2026Review
- Prime editing: Emerging mechanisms, engineering innovations, and next-generation applications.Biodesign research · 2026Review
- Towards precision medicine for brain arteriovenous malformations.The Journal of clinical investigation · 2026Review
- Gene Editing Strategies for Duchenne Muscular Dystrophy: From Molecular Mechanisms to Clinical Translation.Cells · 2026Review
876 more citing papers are in PubMed but not listed here.
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Authors and funding
12 authors.
Funding
Abstract
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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.