Rational Design for Developing Precise Base Editors

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) systems induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs. As a genome engineering tool, CRISPR systems are widely used in biology, biochemistry, and therapy. In this study, we applied CRISPR systems for developing novel methods which are useful for protein production and we developed more precise gene editing tools by engineering CRISPR-based tools. In the first part of the study, by combining DNA cleavage via a CRISPR system and DNA ligation via a Gibson Assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. With this method, we generated DNA plasmids that encode engineered Cas9 variants fused with adenosine deaminases and show that the re-constructed plasmids convert adenosine at target sites containing the non-canonical PAMs. Next, we present a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome. We integrated fluorescent protein in the hotspots by CRISPR systems as a proof-of-concept and show that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. In the next part of the study, we focused on adenine base editor that comprise an adenosine deaminase, evolved in vitro, and a CRISPR-Cas9 nickase. Adenine base editor is an effective gene editing tool that catalyze specific A-to-G conversions at genomic sites of interest without generating a DNA double-strand break or requiring a donor DNA template. Here, we show that ABEs also convert cytosine in a narrow editing window and in a confined TC*N sequence context. ABE-induced cytosine substitutions reduce the number of suitable targeting sites for high specificity base editing. To reduce the cytosine editing activity, we engineered adenosine deaminases by rational design. We found that a D108Q mutation in adenosine deaminases exhibited greatly reduced cytosine deamination activity. In addition, we found that ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool that broadens the utility of base editors.