Transcript-specific induction of stop codon readthrough using CRISPR-dCas13 system

Abstract

AbstractStop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using CRISPR-dCas13 system. Using specific guide RNAs, we targeted dCas13 to the downstream region of the canonical stop codons of mammalianAGO1andVEGFA,which are known to exhibit natural SCR. Results of readthrough assays revealed the enhancement of SCR of these mRNAs (both exogenous and endogenous) caused by dCas13. This effect was associated with ribosomal pausing, which has been reported in several SCR events. Furthermore, our results show that CRISPR-dCas13 can induce SCR across premature termination codons (PTC) in the mRNAs of green fluorescent protein andTP53. Finally, we demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC inHBBmRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.