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

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.

Keywords: ARCA; CRISPR; Cap 1; Cas9; CleanCap; capping; innate immunity; mRNA; uridine depletion.

Graphical abstract

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.

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 mRNA 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.

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.

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.

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.