Cited by 262CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryRNA regulation and disease

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Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification

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Vaidyanathan, Sriram, Azizian, Krist T., Haque, A.K.M. Ashiqul, Henderson, Jordana M., Hendel, Ayal, Shore, Sabrina, Antony, Justin S., Hogrefe, Richard I., Kormann, Michael S.D., Porteus, Matthew H., McCaffrey, Anton P.·2018·Molecular Therapy Nucleic Acids

DOI 10.1016/j.omtn.2018.06.010PMC PMC6076213

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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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Figure 1 Eukaryotic Cap Structures and Cap Analogs (A) Eukaryotic cap structure. Presence of 2′-O-methyl groups at R1 and R2 determine if a cap structure is Cap 0, Cap 1, and Cap 2 as indicated. (B) Structure of anti-reverse cap analog used in standard co-transcriptional capping. (C) Structure of CleanCap AG Cap1 Trimer. (D) Proposed mechanism of CleanCap co-transcriptional initiation in which the AmG dimer portion of CleanCap docks onto the +1 and +2 template nucleotides. Initiation occurs when CleanCap couples to an NTP occupying the +3 position.CC BY · PubMed Central

Figure 2 Indel Formation in CD34+ HSPCs Nucleofected with Modified Cas9 mRNAs CD34+ HSPCs were nucleofected with 3 μg of the indicated Cas9 mRNA and 2 μg IL2RGlocus MS-sgRNA. 6 μg Cas9 RNP complexed to 3.2 μg IL2RGlocus MS-sgRNA was nucleofected for comparison. ARCA 5meC/Ψ is our previously published Cas9 mRNA6 and was also included for comparison. Cells were isolated after 4 days, and indel formation was assessed by TIDE analysis. Bars represent mean ± SEM of at least 5 independent transfections. White and gray bars indicate RNeasy and HPLC-purified mRNAs, respectively. sgRNA complexed to Cas9 RNP was included as a control. ***p < 0.0005 and *p < 0.05.CC BY · PubMed Central

Figure 3 IFN Response Generated by THP-1 Dual Cells Transfected with Modified Cas9 mRNAs THP-1 dual cells were transfected in sextuplicate with 100 ng of the indicated mRNAs complexed with 1 μL transfection reagent mRNA-In. At 24 hr, Lucia expression in the media was assayed as a measure of IFN activity. Bars represent mean ± SEM of three independent assays comprising a total of 18 replicates. *p < 0.05.CC BY · PubMed Central

Figure 4 Amounts of IL-12, TNF-α, and IL-6 in Whole Human Blood Transfected with Modified Cas9 mRNAs To assess immune responses to transfected mRNAs, whole blood from healthy human volunteers (n = 3) was transfected with 10 μg mRNA complexed with 10 μL TransIT (https://www.mirusbio.com/). After 6 or 24 hr of incubation, sera were isolated and (A) IL-12, (B) TNF-α, or (C) IL-6 was measured by ELISA. Bars represent mean ± SEM. *p < 0.05 relative to 6-hr blood-only control.CC BY · PubMed Central

Figure 5 Amounts of IL-12, TNF-α, and IL-6 in the Sera of Mice after Intravenous Infusion of Modified Cas9 mRNAs To assess immune responses in vivo, 20 μg Cas9 mRNA encapsulated in chitosan-coated PLGA nanoparticles was injected intravenously (n = 3) into the tail vein of mice. After 6 or 24 hr of incubation, sera were isolated and (A) IL-12, (B) TNF-α, or (C) IL-6 was measured by ELISA. Blood treated with R-848 serves as a positive control. Bars represent mean ± SEM. *p < 0.05 relative to 6-hr blood-only control.CC BY · PubMed Central

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Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes
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Recognition of 5′ Triphosphate by RIG-I Helicase Requires Short Blunt Double-Stranded RNA as Contained in Panhandle of Negative-Strand Virus
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Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA
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Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation
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High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells
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Activation of MDA5 Requires Higher-Order RNA Structures Generated during Virus Infection
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Viral and cellular mRNA capping: Past and prospects
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“Cap 0 and 50-triphosphate bind RIG-I with similar affinities, while Cap 1 modification abrogates RIG-I signaling.(37) Similarly, Cap 1 prevents detection by MDA5.”
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Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L
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MethodSelf-citation
Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin
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Background
Nucleic acid agonists for Toll‐like receptor 7 are defined by the presence of uridine ribonucleotides
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BackgroundInfluential
MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells
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RNAs containing modified nucleotides fail to trigger RIG-I conformational changes for innate immune signaling
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“Based on this work, many first-generation mRNAs were modified with 5meC and J.46 They showed that chemical modification of mRNA limited TLR signaling,43 decreased activation of 20-50-oligoadenylate synthetase,45 and decreased binding to PKR.44 Durbin et al.47 showed that RNAs modified with m6A bind RIG-I with reduced…”
BackgroundInfluential
RIG-I Forms Signaling-Competent Filaments in an ATP-Dependent, Ubiquitin-Independent Manner
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Background
Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5’ppp- mRNAs
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“Lastly, IFITs sense aberrant cap structures.(31) Therefore, Cas9 mRNA must avoid both endosomal TLRs and cytosolic PRRs to achieve maximal translation and protein activity.”
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Self-coded 3′-Extension of Run-off Transcripts Produces Aberrant Products during in Vitro Transcription with T7 RNA Polymerase
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Innate immune restriction and antagonism of viral RNA lacking 2׳-O methylation
Hyde, J. L., & Diamond, M. S. (2015) · Virology · doi:10.1016/j.virol.2015.01.019 · Cited by 194
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Quantifying on- and off-target genome editing
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Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing
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See all 262

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: Haque, A.K.M. Ashiqul
Henderson, Jordana M.
Hendel, Ayal
Shore, Sabrina
Antony, Justin S.
Kormann, Michael S.D.
Porteus, Matthew H.
McCaffrey, Anton P.
Venue: Molecular Therapy Nucleic Acids
Publisher: Elsevier BV
Published: 2018-09
Volume: 12
Pages: 530-542
ISSN: 2162-2531 (ISSN-L)
DOI: 10.1016/j.omtn.2018.06.010
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: cc-by
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/legal/tdmrep-license (tdm)
creativecommons.org/licenses/by/4.0/ (vor)
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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Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification

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