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- Title
Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro.
- Authors
Fujita, Toshitsugu; Nagata, Shoko; Yuno, Miyuki; Fujii, Hodaka
- Abstract
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.
- Subjects
NUCLEOPROTEINS; REVERSE transcriptase; CRISPRS; NUCLEOTIDE sequence; BACULOVIRUSES
- Publication
Biology Methods & Protocols, 2021, Vol 6, Issue 1, p1
- ISSN
2396-8923
- Publication type
Article
- DOI
10.1093/biomethods/bpab009