Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope

Tingting Jiang,Luke H Rhym,Martin Mense,Wen Xue,Zhiping Weng,Yi Cheng,Anton P Mccaffrey,Qin Wang,Jordana M Henderson,Yueying Cao,Kevin Coote,Daniel G Anderson,Hillary C Valley,Hermann Bihler,David R Liu,Xiao-Ou Zhang,Gregory A Newby

doi:10.1038/s41467-020-15892-8

Abstract

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.

Highlights

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings
When we tested a non-viral delivery method of RA6.3 in vivo, we found that lipid nanoparticle (LNP)-mediated delivery of unmodified mRNA supported three-fold lower editing efficiency compared to plasmid-delivered RA6.3 in Tyrosinemia I mice[4]
Previous reports show that unmodified cytidine base editor mRNA and guide RNA could effectively edit embryos and oocytes[14,15], our data show that unmodified Adenine base editor (ABE) mRNA does not effectively express in HEK293T cells and unmodified guide RNA cannot mediate efficient editing in somatic cell culture

Summary

Introduction

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. We engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo. The therapeutic potential of ABE has been demonstrated by delivery of DNA-encoded base editors to adult animal disease models via plasmids[4] or AAVs9,12,13 These approaches raise the potential for DNA integration or off-target effects due to long-term exposure to the gene editing machinery, hindering their clinical relevance. Our engineered RNA-encoded system expands the application scope of base editors

Methods

Results

Conclusion