Programmable RNA acetylation with CRISPR-Cas13

Recent studies claim that N4-acetylcytidine (ac4C) modification of RNA confers crucial regulatory roles, such as increasing translation efficiency and prolonging its half-life. However, the absence of methods for selectively acetylating specific RNA molecules hampers linking ac4C to cell physiology. Here, we developed an efficient molecular tool that incorporates ac4C on a specific transcript of interest. Through protein engineering, we developed a hyperactive variant of N-acetyltransferase 10 (NAT10), designated eNAT10. When fused to the programmable RNA-targeting protein dCas13, eNAT10 enables robust acetylation of various target RNAs in multiple contexts. RNA acetylation by dCas13-eNAT10 was highly dependent on co-transfected guide RNA, highlighting its specificity. We also describe the first programmable RNA chemical modification in vivo using dual-AAV. Utilizing our system, we found that acetylation of RNA may modulate the subcellular localization of modified transcripts. We anticipate that our tool will facilitate numerous studies on ac4C functions across different cellular and disease contexts. Overall design: acRIP-seq & ac4C-seq profiling of off-target RNA acetylation in HEK293T cells transfected with dCas13-eNAT10, at 2-days post-transfection. acRIP-seq profiling of off-target RNA acetylation in mouse liver tissue transduced with dCas13-eNAT10 encoding AAV, at 3-weeks post-transduction. RNA-seq profiling of HEK293T cells transfected with dCas13-eNAT10, at 2-days post-transfection. Nuclear-cytoplasmic fractionation-seq profiling of HEK293T cells transfected with dCas13-eNAT10, at 2-days post-transfection.