RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Nat Commun. 2018 Jul 6;9(1):2641. doi: 10.1038/s41467-018-05073-z.
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.
基于 RNA 的药物依赖于化学修饰来提高体内的效力和降低免疫原性。化学修饰也可能改善 CRISPR-Cas9 治疗相关的向导 RNA。Cas9 同源物是 RNA 指导的微生物效应物，可切割 DNA。在这里，我们探索了 crRNA 向导和 tracrRNA 辅助因子所有位置的化学修饰。我们鉴定了几种比其未修饰对应物更有效的大量修饰的 crRNA 和 tracrRNA。此外，我们还描述了完全化学修饰的 crRNA 和 tracrRNA（不含 2'-OH 基团），它们在人细胞中具有功能。这些设计将有助于基于 Cas9 的治疗，因为大量修饰的 RNA 在体内往往更稳定（从而提高效力）。我们预计我们的设计将通过 RNP 和 mRNA 递送来改善 Cas9 的体内和体外用途。
