Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

Shuo Li,Xing-Ming Zhao,Bo Yuan,Xiaolin Wang,Jixin Cao,Tian-Lin Cheng,Jiayi Qiu,Zilong Qiu,Jinlong Chen,Jingqi Chen

doi:10.1038/s41467-020-19730-9

Abstract

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.

Highlights

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications
Existing adenine base editors (ABEs) tools were initially developed by fusing TadA-TadA* heterodimer to the N-terminus of nCas[9] (D10A), in which TadA served as a scaffold for substrate without single-stranded DNA (ssDNA) deamination activity while TadA* is an evolved adenosine deaminase with obvious ssDNA deamination activity
We generated a series of functional ABE variants with diversified editing scopes and minimized RNA off-target activities using domain insertion profiling, which confirmed that domain insertion is an effective and powerful strategy for the development of Cas9-based fusion proteins

Summary

Introduction

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. Diversified editing scopes will enable ABEs to access more target nucleotides and expand their capabilities for the correction of disease-associated SNPs. As single-stranded DNA (ssDNA) substrates are required for effective editing, the accessibility of ssDNA loop to deaminases is critical for the diversification of editing scopes, in addition to the features of deaminases and protospacer adjacent motif (PAM) of CRISPR-Cas systems[3,5,6,7,8,9,10]. Though ABE variants with minimized RNA off-target activities have been developed through deaminase engineering, their editing scopes were severely restricted, with editing windows mainly at positions 4–8 (the PAM was counted as 21–23 unless otherwise indicated). The relative distances of docking sites (DSs) inside Cas[9] to ssDNA loop are more variable as compared to N-/C-termini, and domain insertion inside Cas[9] may limit the excessive flexibility of functional domains, so it is possible that domain insertion could achieve scope diversifications and off-target minimization simultaneously

Methods

Results

Conclusion