Streptococcus thermophilus CRISPR-cas9 systems enable specific editing of the human genome
Open accessAbstract
RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.
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St1Cas9 and St3Cas9 from Streptococcus thermophilus recognize longer PAM sequences, which are 5'-NNAGAAW-3' and 5'-NGGNG-3', respectively.
“Similarly, St1Cas9 and St3Cas9 from Streptococcus thermophilus recognize longer PAM sequences, which are 5’-NNAGAAW-3’ and 5’-NGGNG-3’, respectively (Muller et al., 2016).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Muller et al., 2016
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- Authors
- Müller, M., Lee, C. M., Gasiunas, G., Davis, T. H., Cradick, T. J., Siksnys, V., Bao, G., Cathomen, T., & Mussolino, C.
- Affiliations
- University of Freiburg (DE)
- Georgia Institute of Technology (US)
- Corresponding author
- Cathomen, Toni
- Mussolino, Claudio
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2016-03
- Volume
- 24
- Issue
- 3
- Pages
- 636-644
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC4786917
- OpenAlex
- W2221514390
- Cited by
- 254
- References
- 57
- Citations by year
- 2026: 9 · 2025: 18 · 2024: 21 · 2023: 23 · 2022: 38 · 2021: 36 · 2020: 36 · 2019: 17 · 2018: 18 · 2017: 22 · 2016: 16
- FWCI
- 8.10
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (PN2EY018244)
- Bundesministerium für Bildung und Forschung (01EO0803)
- 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
- SpCas9, CRISPR/Cas9 genome editing, off-target mutagenesis, protospacer adjacent motif, PRKDC, CARD11, human genome editing, gene therapy
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Base Sequence, Binding Sites, Cell Line, Endonucleases, Enzyme Activation, Genetic Vectors, Humans, Protein Binding, Substrate Specificity, Genome, Human, Streptococcus thermophilus, CRISPR-Cas Systems
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