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.

Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases

“…229 In mRNA, only a small subset of these naturally occurring modifications is reported to be essential for reducing innate immune response and improving mRNA expression and stability. 215,230,231 N6-methyladenosine (m6A) is one of the most frequent modifications in eukaryotic mRNA. Insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) preferentially recognize m 6 A mRNA and guard the modified mRNA against decay.…”

“…231,240 Cas9 activity has been reported to produce significantly higher insertion or deletion (indel) and to be less immunogenic when uridine depletion has been used with 5-methoxyuridine (5moU) modification compared to unmodified and J-UTPmodified Cas9 mRNA. 215 Pharmacologically favorable mRNA that has undergone modification and sequence optimization still needs to be transferred in lung using carriers, to circumvent the naturally occurring barriers the lung possesses.…”

New Bicistronic TALENs Greatly Improve Genome Editing

“…Our design produces mRNAs that do not activate, or only minimally activate, these RNA‐sensing pathways. Exogenous mRNA can activate innate immunity pathways through the following mechanisms: Pattern‐recognition receptors (PRRs), present in both endosomes and the cytosol (Vaidyanathan et al., ); Retinoic‐acid‐inducible gene I (RIG‐I), which recognizes 5′ triphosphates (5′ppp; Hornung et al., ; Pichlmair et al., ) or diphosphates (Goubau et al., ), panhandle structures of viral genomic RNA (Schlee et al, ; Weber & Weber, ), and uridine‐rich sequences (Chiang et al, ; Uzri & Gehrke, ); Melanoma differentiation‐associated protein 5, (MDA5) activated by binding very long double‐stranded RNA (dsRNA; Feng et al., ); Protein kinase R (PKR), dsRNA stretches of at least 33 nucleotides (Hull & Bevilacqua, ); Interferon (IFN)‐induced tetratricopeptide repeat (IFIT) proteins, which sense aberrant cap structures (Kumar et al., ); Pathogen‐associated molecular patterns associated with exogenous RNA (viral or transfected RNA) that activate Toll‐like receptors (TLRs), which recognize single‐stranded RNA, (ssRNA) and dsRNA (Alexopoulou, Holt, Medzhitov, & Flavell, ; Heil et al., ); …”

“…• Pattern-recognition receptors (PRRs), present in both endosomes and the cytosol (Vaidyanathan et al, 2018);…”

“…To escape these problems, mRNA must avoid both endosomal TLRs and cytosolic PRRs, include cap structure modifications, chemical modifications, and sequence engineering (uridine depletion), or even undergo a high-performance liquid chromatography purification step (Vaidyanathan et al, 2018):…”