Abstract
In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.
Highlights
Modulation of gene expression or alteration of gene transcripts provides a critical layer of regulation at the RNA level in living cells
The results indicated that the dCas13a-hADAR2d system was capable of editing reporter mRNA at a specific site, and both CRISPR RNA (crRNA) and pairing RNAs (pRNAs) were required for editing activity
The tdh1 and nmt1 genes displayed comparable transcript abundance (560 and 340 mRNA molecules per cell, respectively) [40], tdh1 and nmt1 exhibited dramatically different editing efficiencies, of 54.5 and 0%, respectively. These results indicated that the dCas13a-hADAR2d system was applicable to a broad range of genes for RNA editing
Summary
Modulation of gene expression or alteration of gene transcripts provides a critical layer of regulation at the RNA level in living cells. Members of the Cas family display the unique ability to target single-strand RNA. One member of the Cas family, Cas13a from Leptotrichia shahii (LshCas13), was found to knockdown target transcripts in Escherichia coli and mammalian cells and has been used in nucleic acid detection and RNA tagging [7,8,11,12]. Another member of the Cas family, Cas13b from Prevotella sp. P5-125, was employed to construct a fusion protein for targeted editing of gene transcripts in mammalian cells, which was shown to correct disease-associated mutations in human cell lines [10]
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