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Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants
Open accessAbstract
A PAMless base editor: CRISPR-Cas DNA base editing typically requires a specific motif for targeting known as a protospacer-adjacent motif (PAM). This requirement limits the sequences within a genome that can be targeted. Walton et al. engineered specific variants of the Streptococcus pyogenes Cas9 enzyme named SpG and SpRY that could recognize and edit a wider array of PAMs. Using SpRY, the authors were able to correct previously uneditable mutations associated with human disease. Although off-target effects were observed for these engineered Cas enzymes at levels similar to those of the wild-type enzyme, depending on the context, these engineered enzymes widen the potential applications of precision genome editing. Science , this issue p. 290
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“Efforts are ongoing to develop PAM-free Cas9 molecules to eliminate PAM site constraints altogether, thereby increasing the flexibility and effectiveness of genome editingthrough structure-based rational engineering or by altering amino acid substitutions in the PAM-interacting domain (Walton et al., 2020).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Walton et al., 2020
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“CRISPR-Cas dominates the field because of the ease of using guide RNAs directing the CRISPR complex to specific DNA sequences and the increasing ability to target most sites in the genome.121,122”
From Fetal hemoglobin in sickle cell anemia (2020) · cites this paper as [122]
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“Building on this, Walton et al. (2020) introduced the SpG variant, which can recognize a broader range of NGN PAMs.”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Walton et al., 2020
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“These can be targeted in combination with evolved Cas9 variants such as SpCas9-NG20 or SpG.19”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [19]
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“These can be targeted with SpRY.19”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [19]
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“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 [21]
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Metadata
- Authors
- Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P.
- Affiliations
- Massachusetts General Hospital (US)
- Harvard University (US)
- Corresponding author
- Kleinstiver, Benjamin P.
- Venue
- Science
- Published
- 2020-04-17
- Volume
- 368
- Issue
- 6488
- Pages
- 290-296
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC7297043
- OpenAlex
- W3013504322
- Semantic Scholar
- 84609b61a82a11e3122a81b5bdae20a2822e577b
- Cited by
- 1,233
- References
- 48
- Citations by year
- 2026: 157 · 2025: 200 · 2024: 190 · 2023: 213 · 2022: 203 · 2021: 177 · 2020: 93
- FWCI
- 56.07
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Heart, Lung, and Blood Institute (P01-HL142494)
- National Cancer Institute (R00-CA218870)
- American Society of Gene and Cell Therapy
- Margaret Q. Landenberger Research Foundation
- 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, SPG, SpRY, base editing, genome editing, human cells
- MeSH terms
- Protein Domains, Gene Editing, CRISPR-Associated Protein 9, Humans, Substrate Specificity, Mutagenesis, Gene Targeting, Genetic Predisposition to Disease, HEK293 Cells, CRISPR-Cas Systems
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