GSE184244
CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo
RNA base editing represents a promising alternative for genome editing. Recent approaches harness the endogenous RNA editing enzyme ADAR to circumvent problems related to the ectopic expression of an editing enzyme, but they suffer from sequence restriction, lack of efficiency, and bystander editing. Here, we present in-silico optimized CLUSTER guide RNAs, which bind their target mRNAs in a multivalent fashion and thereby enable editing with unprecedented precision as shown by next generation sequencing. CLUSTER guide RNAs can be genetically encoded and manufactured into viruses to work in various cell lines. They achieve on-target editing on endogenous transcripts like GUSB and NUP43 with yields up to 45% without bystander editing and have been shown to recruit endogenous ADAR in vivo. The CLUSTER approach tremendously enlarges the sequence space available for guide RNA design and opens new avenues for drug development in the field of RNA base editing. Overall design: Identification of off-target editing events and gene expression changes in HEK293FT cell line transfected with guide RNA (gRNA) for RNA editing by RNA-Seq, 2 samples (Cluster gRNA and Leaper gRNA) , 1 control sample (NT gRNA), 2 biologically independent experiments for each sample, 6 samples in total