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

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.

Comparison of the efficiency and precision of Base editor and CRISPR/Cas9 for inducing defined point mutation (S395F) in ovine embryos

“…The cytosine deaminase APOBEC1 fused to the BE4-Gam system can induce single nucleotide variants(SNVs) in the absence of sgRNA (Komor et al, 2016). WGS was used to scan the SNVs caused by BE3 and BE4 in mouse embryos, and the results demonstrated an approximately 2-fold increase in the number of off-target mutations in BE4-edited mouse embryos (Fan et al, 2021), and ≥20-fold increase in BE3-edited mouse embryos (Zuo et al, 2019). In contrast, CRISPR/Cas9 does not introduce an excess of off-target mutations independent of sgRNAs (Iyer et al, 2018;Willi et al, 2018).…”

Challenges of Gene Editing Therapies for Genodermatoses

“…Their unpredictable nature poses a great challenge, as the detection of singlenucleotide mutations caused by deaminases requires costly whole-genome sequencing and the careful interpretation of obtained data. Both CBE and ABE have also been shown to extensively deaminate nucleotides in RNA molecules, independently of DNA changes, and to cause considerable differences in gene expression and splicing [95][96][97]. Several modified deaminases have been developed to combat Cas9-independent off-target mutations: SECURE, RrA3F, AmAPOBEC1, PpAPOBEC1 and SsAPOBEC3B for cytosine base editors and SECURE-ABE for ABEs [96,98,99].…”

Acceleration of the Deamination of Cytosine through Photo-Crosslinking

“…However, there are possibilities of off-target effects [ 17 ]. In addition, there are reports of other enzymatic methods such as RNA-specific adenosine [ 18 ] and cytosine deaminases [ 19 ], along with chemical methods such as bisulfite conversion [ 20 ]; however, they also have some limitations such as non-specific deamination and non-applicability for in vivo applications.…”