Programmable RNA acetylation with CRISPR-Cas13

Prof. Won Do Heo’s group at KAIST has engineered a CRISPR–Cas13–based system that installs N4acetylcytidine (ac4C) on RNA transcripts of interest in cultured cells and in live mice, enabling causal, functional study of RNA acetylation. By fusing catalytically dead Cas13 (dCas13) to an engineered, hyperactive NAT10 variant (eNAT10), the team achieved robust and selective acetylation of target RNAs and uncovered a distinct function of ac4C in regulating subcellular RNA localization. ac4C is an abundant RNA modification written by an enzyme named NAT10, yet its prevalence and function on human mRNA have been debated due to reliance on global NAT10 perturbations that confound transcriptlevel effects. The team addressed this gap with a programmable, guide RNA–directed acetylation platform that operates on specific transcripts without broadly altering the whole transcriptome. Systematic truncation and engineering of NAT10 yielded eNAT10, which, when fused to dCas13, potentiated ontarget acetylation while maintaining specificity across diverse cellular contexts. The authors validated the tool through multiomics profiling, including acRIPseq, ac4Cseq, RNAseq, and quantitative proteomics, and confirmed efficient, targeted ac4C installation and minimal offtarget perturbation. Singlenucleotide mapping on engineered targets revealed a strong preference for the previously reported 5′CCG3′ sequence motif. In addition, motifdisrupting mutations markedly reduced acetylation efficiency, highlighting sequence rules that guide NAT10mediated writing. As expected for codingregion ac4C, the team observed enhanced translation of acetylated mRNAs. A key discovery emerged from a nucleartargeted version of the platform that acetylates newly transcribed RNAs: acetylated transcripts were enriched in the cytoplasm and depleted in the n...read more