Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing

Jiao Fan,Chao Ren,Yige Ding,Wenjie Shu,Ziguo Song,Xiaolong Wang,Chenchen Du,Chao Li,Jie Yuan,Qiuzhen Chen

doi:10.1038/s42003-021-02406-5

Abstract

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

Highlights

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks
Recent studies have shown that both CBEs and ABEs can induce extensive transcriptome-wide deamination of cytosine or adenosine in human cells; this leads to the generation of thousands of off-target RNA single nucleotide variations (SNVs)[9,10,11,12,13]
To evaluate the deaminase effects of base editors, an RNA-seq based analysis of transcriptome-wide Differentially expressed genes (DEGs) was conducted. This was applied to one type of CBE (BE3; APOBEC1-nCas9-UGI) and one type of ABE (ABE7.10; TadA-TadA*-nCas9), combined with GFP and either with or without a single guide RNA in HEK293T cells

Summary

Introduction

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks Both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Transcriptome-wide differentially expressed genes (DEGs) and differential alternative splicing (DAS) events that had been induced by CBEs or ABEs were identified Both cytosine and adenine base editors had generated hundreds of DEGs and DAS events. Engineering of CBE or ABE variants had only negligible effects on the prevention of DEG and DAS occurrences and even increased the emergence of DEGs and DAS events because of the overexpression of cytosine and adenine deaminases These data highlight a previously unreported aspect of deaminase effects on gene expression and splicing, which apparently cannot be eliminated by genetic engineering. These results have important implications for the future use of base editors in both research and as therapeutic agents, highlighting a full characterization of deaminase enzyme activities in base editors

Methods

Results

Conclusion