Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for genome editing because of its ability to cleave specific DNA sequences. Recently, RNA-specific CRISPR systems have been reported. CRISPR systems, consisting of a guide RNA (gRNA) and a nuclease-dead form of Cas13a (dCas13a), can be used for RNA editing and visualization of target RNA. In this study, we examined whether a recombinant CRISPR/dCas13a ribonucleoprotein (RNP) complex could be used to inhibit reverse transcription (RT) in a sequence-specific manner in vitro. Recombinant Leptotrichia wadei dCas13a was expressed using the silkworm-baculovirus expression system and affinity-purified. We found that the CRISPR/dCas13a RNP complex, combined with a chemically synthesized gRNA sequence, could specifically inhibit RT of EGFR and NEAT1, but not nonspecific RNA. Thus, the CRISPR/dCas13a RNP complex can inhibit RT reactions in a sequence-specific manner. RT inhibition by the CRISPR/dCas13a system may be useful to assess target binding activity, to discriminate RNA species retaining target sequences of gRNA, or to suppress RT from undesirable RNA species.