RT Journal Article
SR Electronic
T1 Implementation of CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 284661
DO 10.1101/284661
A1 Xinyun Jing
A1 Bingran Xie
A1 Longxian Chen
A1 Niubing Zhang
A1 Yiyi Jiang
A1 Hang Qin
A1 Pei Hao
A1 Sheng Yang
A1 Xuan Li
YR 2018
UL http://biorxiv.org/content/early/2018/03/19/284661.abstract
AB In contrast to genome editing that introduces genetic changes at DNA level, disrupting or editing genes’ transcripts provides a distinctive approach to perturb a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulation of RNA, we first implemented a member of 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. While it was shown to knock down targeted endogenous genes’ transcripts, differently from previous studies in E. coli, no collateral cleavage of other non-specific RNA by activated Cas13a-crRNA complex was detected in fission yeast. Second, we engineered a RNA-editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human Adenosine Deaminase Act on RNA 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized the system parameters using a dual-florescence reporter and demonstrated its utility in editing of randomly selected endogenous genes’ transcripts. Our engineered RNA-editing system enables a new toolset for transcriptomic manipulation that is widely applicable in basic genetic and biotechnological research.