Heart-specific gene silencing via single-AAV-delivered base editing [RNA-Seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE265935 | GEO | 2025/01/21
REPOSITORIES: GEO
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Project description:To develop a new therapeutic strategy for cardiac ischemia/reperfusion injury, we ablated the oxidative activation site of CaMKIIδ in the heart using CRISPR-Cas9 adenine base editing technology. Adult C57Bl6 mice were subjected to control surgery (IR-Sham, 3 biological replicates) or ischemia/reperfusion injury with either no injection (IR, 3 biological replicates), injection of a control virus (IR-Virus control, 3 biological replicates) or injection of a functional CaMKIIδ editing system (IR-Edit, 4 biological replicates). Five weeks after the surgery, hearts were harvested for RNA isolation and subsequent bulk RNA sequencing and gene expression profiling analysis.
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Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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