Abstract
RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2′-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.
Highlights
RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo
Engineered CRISPR systems have the potential to transform the treatment of inherited diseases via genome editing-based cures
Safe, effective, and target-tissue-specific delivery of CRISPR effector proteins and their small RNA guides represents a major barrier to clinical application
Summary
RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. We describe fully chemically modified crRNAs and tracrRNAs (containing no 2′-OH groups) that are functional in human cells These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo ( increasing potency). The crRNA contains a guide sequence that directs the Cas[9] RNP to a specific locus via base pairing with the target DNA to form an Rloop. This process requires the prior recognition of a protospacer adjacent motif (PAM), which for SpyCas[9] is NGG. The tracrRNA consists of an anti-repeat region that pairs with the crRNA, and includes three stem-loops All of these secondary structure elements are required for efficient editing in mammalian systems[9]. Unmodified RNAs are subject to rapid degradation in circulation and within cells[10,11]
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