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

Genome-wide discovery of the impact of cytosine and adenine deaminases on transcriptome-wide gene expression and splicing induced by CBEs and ABEs

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Recently, a varity of cytosine and adenine base editors (CBEs and ABEs) that combines cytosine or adenosine deaminases with CRISPR-Cas9, has been developed. This variety enables highly efficient and precise targeted C-to-T or A-to-G base conversions1-4. Previous studies have reported the induction of unexpected off-target DNA editing in mammalian cells by both CBEs and ABEs. This limited their broad use in biomedical studies and therapeutic applications3,5. Adeno-associated viruses (AAVs) are the most commonly utilized delivery system for gene therapies. The main reason is their efficiency of DNA editing, as well as their capability to maintain long-term gene expression without toxicity or immune responses to the viral vector in vivo6-8. Thus, the extent of the potential effects of DNA base editor induced deaminases is of great importance. Recent studies have shown that both CBEs and ABEs can indeed 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)9-13. Whether the presence of this large number of unwanted RNA SNVs has genetic or transcriptional consequences has not been comprehensively explored to date, this lack of data may lead to underestimation of the effect. Although engineering-improved CBEs or ABEs can largely decrease the number of off-target RNA SNVs in human cells11,12, a comprehensive characterization of the effects of CBE or ABE deaminases on RNA is crucial to fully realize their utility. 

This study analyzed RNA-seq datasets from two recent studies9,12. 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. However, these phenomena had previously not been reported. Furthermore, 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, thus highlighting for a full characterization of deaminase enzyme activities in base editors.

Figure 1. Identification and characterization of transcriptome-wide DEGs and DASs induced by DNA base editors. 

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Wenjie Shu

PI, Department of Biotechnology, Beijing Institute of Radiation Medicine