Post-transcriptional cleavage and polyadenylation of messenger and long noncoding RNAs is coordinated by a supercomplex of ~20 individual proteins within the eukaryotic nucleus1,2. Polyadenylation plays an essential role in controlling RNA transcript stability, nuclear export, and translation efficiency3–6. More than half of all human RNA transcripts contain multiple polyadenylation signal sequences that can undergo alternative cleavage and polyadenylation during development and cellular differentiation7,8. Alternative cleavage and polyadenylation is an important mechanism for the control of gene expression and defects in 3’ end processing can give rise to myriad human diseases9,10. Here we show that fusion of catalytically dead Cas13 to a single mammalian polyadenylation factor, Nudix hydrolase 21 (NUDT21), allows for site-specific CRISPR-Cas13-guided cleavage and polyadenylation of RNA in mammalian cells. This approach, which we named Postscriptr, can be utilized for the non-genomic manipulation of gene expression and may have potential future therapeutic applications for treating human RNA processing diseases.
Human monogenetic diseases can arise from the aberrant expansion of tandem nucleotide repeat sequences, which when transcribed into RNA, can misfold and aggregate into toxic nuclear foci 1 . Nuclear retention of repeat-containing RNAs can disrupt their normal expression and induce widespread splicing defects by sequestering essential RNA binding proteins. Among the most prevalent of these disorders is myotonic dystrophy type 1 (DM1), a disease occurring from the expression of a noncoding CTG repeat expansion in the 3'UTR of the human dystrophia myotonica protein kinase (DMPK) gene 2,3 . Here we show that RNAbinding CRISPR-Cas13, with a robust non-classical nuclear localization signal, can be efficiently targeted to toxic nuclear RNA foci for either visualization or cleavage, tools we named hilightR and eraseR, respectively. HilightR combines catalytically dead Cas13b (dCas13b) with a fluorescent protein to directly visualize CUG repeat RNA foci in the nucleus of live cells, allowing for quantification of foci number and observation of foci dynamics. EraseR utilizes the intrinsic endoribonuclease activity of Cas13b, targeted to nuclear CUG repeat RNA, to disrupt nuclear foci. These studies demonstrate the potential for targeting toxic nuclear RNA foci directly with CRISPR-Cas13 for either the identification or treatment of DM1. The efficient and sequence programmable nature of CRISPR-Cas13 systems will allow for rapid targeting and manipulation of other human nuclear RNA disorders, without the associated risks of genome editing.Myotonic dystrophy type 1 (DM1) is an autosomal dominant human monogenic disease characterized by progressive myotonia, muscle wasting, cardiac arrhythmias, and cognitive dysfunction 4 . DM1 is the most common form of adult-onset muscular
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