The DOI matches its Crossref record; 69 of the 69 references it cites were found in a registry or an index.
Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification
Open accessAbstract
The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.
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“Others strategies aim to impair the PRR recognition of IVT mRNA with the use of modified nucleosides,16 uridine depletion in the coding sequence,17 or dephosphorylation of 5′PPP mRNAs.18”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [17]
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“dsRNA can activate PRR such as TLR3,52,156 whereas ssRNA activates TLR7 and TLR8 to produce IFN-I, thus inducing inflammation in the host and interrupting mRNA translation.157,158”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [157]
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“TLR7 and TLR8 receptors bind to guanosine- or uridine-rich regions in mRNA and trigger the production of type I interferons, such as IFNα, that can block mRNA translation24.”
From mRNA vaccines for infectious diseases: principles, delivery and clinical translation (2021) · cites this paper as [24]
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Metadata
- Authors
- Vaidyanathan, S., Azizian, K. T., Haque, A. K. M. A., Henderson, J. M., Hendel, A., Shore, S., Antony, J. S., Hogrefe, R. I., Kormann, M. S. D., Porteus, M. H., & McCaffrey, A. P.
- Affiliations
- Stanford Medicine (US)
- TriLink BioTechnologies (United States) (US)
- University of Tübingen (DE)
- Bar-Ilan University (IL)
- Corresponding author
- McCaffrey, Anton P.
- ORCID
- Venue
- Molecular Therapy Nucleic Acids
- Publisher
- Elsevier BV
- Published
- 2018-09
- Volume
- 12
- Pages
- 530-542
- ISSN
- 2162-2531 (ISSN-L)
- PMC
- PMC6076213
- OpenAlex
- W2810403368
- Cited by
- 262
- References
- 69
- Citations by year
- 2026: 19 · 2025: 34 · 2024: 45 · 2023: 47 · 2022: 48 · 2021: 40 · 2020: 21 · 2019: 7
- FWCI
- 6.05
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Nucleic Acids (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- School of Medicine, Stanford University
- Amon G. Carter 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 Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- Cas9 mRNA, pseudouridine, innate immune response, immunogenicity, genome editing, HPLC purification, double-stranded RNA, CD34+ cells, cytokine response
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