ArticleMolecular therapy. Nucleic acids2018
Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification.
Article in Molecular therapy. Nucleic acids, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 149 papers.
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Who cites it
149 citing papers in PubMed, 260 citations in OpenAlex.
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- Immunology of RNA-based vaccines: The critical interplay between inflammation and expression.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Review
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- Lipid Nanoparticle Development for A Fluvid mRNA Vaccine Targeting Seasonal Influenza and SARS-CoV-2.NPJ vaccines · 2025Article
- Multiplex base editing to protect from CD33 directed drugs for immune and gene therapy.Nature communications · 2025Article
89 more citing papers are in PubMed but not listed here.
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Authors and funding
11 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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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