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

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

doi:10.1016/j.omtn.2018.06.010

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.

Highlights

The Cas9/guide RNA (Cas9/gRNA) system, which is derived from the type II bacterial CRISPR adaptive immune system, is a powerful tool for manipulating genomes.[1,2,3,4] The Cas9/gRNA system consists of an RNA-guided nuclease (Cas9) and a single short gRNA
Our work suggests that uridinedepleted Cas[9] mRNA modified with 5-methoxyuridine or pseudouridine may be optimal for the broad use of Cas[9] both in vitro and in vivo
Synthetic mRNAs are co-transcriptionally capped by including a cap analog in excess in the transcription reaction

Summary

Introduction

The Cas9/guide RNA (Cas9/gRNA) system, which is derived from the type II bacterial CRISPR adaptive immune system, is a powerful tool for manipulating genomes.[1,2,3,4] The Cas9/gRNA system consists of an RNA-guided nuclease (Cas9) and a single short gRNA. Various methods have been described for delivery of the Cas[9] protein into the nucleus These include expression of Cas[9] protein from a plasmid[6] or viral vectors,[7] transfection of recombinant Cas[9] protein complexed to a gRNA (ribonucleoprotein or ribonucleoprotein [RNP] complex),[6,8,9] or expression from a transfected Cas[9] mRNA.[6] Expression of Cas[9] protein from a plasmid or viral vector may be problematic because it risks integration of the promoter and/or Cas[9] gene cassette at the double-stranded break site, a feature of all double-stranded DNA vectors, or random integration of the DNA vector into the genome.[10] By way of contrast, Cas[9] protein and mRNA do not pose the risk of Cas[9] gene integration, and they induce limited off-target effects due to transient expression.[5,11,12] nanoparticle delivery of Cas[9] protein has been reported, the most common approach to deliver transgenes into cells in vivo involves the use of mRNAs complexed with nanoparticles. This makes Cas[9] mRNA an attractive tool for genome editing in hard-to-transfect cells or tissues

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