AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects

Mingming Liang,Liangxue Lai,Zhiquan Liu,Hongmei Liu,Mao Chen,Tingting Sui,Zhanjun Li,Huanhuan Shan

doi:10.3390/cells9081786

Abstract

The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly “shut off” nuclease activity of both CBE and ABE systems.

Highlights

The discovery of the CRISPR system, a widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules [1,2,3], has led to a major shift in the fields of biotechnology and molecular biology, including genome editing and gene expression regulation in living cells and organisms [4,5]
Our results show that AcrIIA5 suppresses base editors and reduces their off-target effects in mammalian cells efficiently
The results show that the off-target effects of BE3 and ABE7.10 were significantly

Summary

Introduction

The discovery of the CRISPR system, a widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules [1,2,3], has led to a major shift in the fields of biotechnology and molecular biology, including genome editing and gene expression regulation in living cells and organisms [4,5]. The unexpected off-target effects will restrict the applications of base editors to precision gene therapy in the future [24]

Methods

Results

Conclusion