Cited by 1,100CRISPR and Genetic EngineeringRNA regulation and diseaseCAR-T cell therapy research

Engineered CRISPR-Cas9 nuclease with expanded targeting space

Open access
Nishimasu, Hiroshi, Shi, Xi, Ishiguro, Soh, Gao, Linyi, Hirano, Seiichi, Okazaki, Sae, Noda, Taichi, Abudayyeh, Omar O. +13 more·2018·Science

Abstract

Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

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  • SpCas9-NG and SpG can target NGN PAMs.

    “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 [20]

    The paper reports that SpCas9-NG edited sites with NGA, NGT, NGG, and NGC PAMs, although activity was lower at NGC sites (section labelled Abstract; page not provided). Our copy does not mention SpG, so it supports only the SpCas9-NG part of the claim. (LLM, conf=0.88)

    Experimental — accuracy not yet measured
  • A rationally engineered SpCas9 variant (SpCas9-NG) generated based on the reduced interaction between the third nucleobase of PAMs and the gRNA-Cas9 complex could edit the target site with NG PAMs.

    “A rationally engineered SpCas9 variant (SpCas9-NG) generated based on the reduced interaction between the third nucleobase of PAMs and the gRNA-Cas9 complex could edit the target site with NG PAMs.”
  • 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.”

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Authors
Nishimasu, H., Shi, X., Ishiguro, S., Gao, L., Hirano, S., Okazaki, S., Noda, T., Abudayyeh, O. O., Gootenberg, J. S., Mori, H., Oura, S., Holmes, B., Tanaka, M., Seki, M., Hirano, H., Aburatani, H., Ishitani, R., Ikawa, M., Yachie, N., Zhang, F., & Nureki, O.
Affiliations
  • The University of Tokyo (JP)
  • Broad Institute (US)
  • Keio University (JP)
  • Research Institute for Microbial Diseases, The University of Osaka (JP)
  • Research Center for Advanced Science and Technology, The University of Tokyo (JP)
  • Institute of Medical Science, The University of Tokyo (JP)
Corresponding author
  • Nishimasu, Hiroshi
  • Nureki, Osamu
Venue
Science
Published
2018-09-21
Volume
361
Issue
6408
Pages
1259-1262
ISSN
  • 0036-8075 (ISSN-L)
  • 1095-9203
OpenAlex
W2889513238
Cited by
1,100
References
25
Citations by year
2026: 81 · 2025: 113 · 2024: 143 · 2023: 134 · 2022: 167 · 2021: 169 · 2020: 156 · 2019: 125 · 2018: 11
FWCI
41.07
Type
journal article
Language
en
Open access
Locations
  • Science (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
  • PMC
Funders
Japan Agency for Medical Research and Development
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, crystal structure, indels, C-to-T conversion, activation-induced cytidine deaminase, targeted mutagenesis
MeSH terms
Gene Editing, CRISPR-Associated Protein 9, Bacterial Proteins, Endonucleases, Humans, Protein Engineering, Crystallography, X-Ray, HEK293 Cells, CRISPR-Cas Systems

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