CrispR-Cas13a-mediated manual machine for accurate fixed-point RNA editing.

On May 31st, the international academic journal Nucleic Acids Research published an online research paper entitled Implementation of CRISPR-Cas13a system in fission yeast and its repurposing for RNA editing, co-authored by the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences/Institute of Plant Physiology and Ecology.
the work, the work reported on an artificial machine that uses the newly discovered Cas13 protein (VI type) and its guiding RNA target binding to specific sequence single-stranded RNA, enabling precise fixed-point RNA (A-I) editing by designing and combining it with human-derived RNA desalysase catalytic domain (hADAR2d).
unlike in recent years, when DNA editing is used to modify dna editing(e.g. Cas9) to modify gene/regulatory gene expression, the Cas13 system does not involve changes in encoded gene DNA, but rather editing gene transcription products (mRNAs) to provide new ways and ideas for changing gene function and regulatory expression.
has been a huge success in recent years in the editing of genetic DNA in biological species, including cutting and base replacement, with the use of CRISPR technology, as well as technical limitations.
complements DNA editing technology, a precise fixed-point editing technology for RNA with unique technical advantages and applications.
RNA fixed-point editing does not alter the encoded gene itself (dna base changes cannot be reversed) and is more controllable in time, space and efficiency than DNA editing.
at the same time, RNA editing can be more efficient by modifying multiple genes at the same time and targeting all transcription products of multi-copy genes at the same time, especially for multi-polypheels.
has important application value in the field of gene therapy for diseases, using RNA fixed-point editing to repair mutant genes in disease tissue.
in order to achieve the artificial machine of precise fixed-point RNA editing, Li Xuan research team, in collaboration with researcher Yang Wei, researcher of the Shanghai Pasteur Institute of the Chinese Academy of Sciences, and Wang Hongbing, a professor at the University of Michigan, Usain Tin, have discovered that Cas13a, which has the ability to combine unique sequence single-stranded RNA in the newly discovered CRISPR family, first realized its target endogenous RNA and degradation target RNA functionin in the model bio-split yeast (Spombe).
further, the design uses Cas13a's retrofit product dCas13a (a mutation that loses RNA cutting activity) to fuse dCas13a with a human-derived RNA adenine desalysase catalytic domain (hADAR2d) and introduce it into the split yeast;
to optimize the design and parameters of RNA fixed-point editing machines (editing base position, distance, guiding RNA structure, length, etc.), the researchers built a series of different designs and used a dual fluorescence reporting system to test the editing efficiency of different settings, resulting in optimal parameters for precision RNA editing machines.
the optimized editing machine achieved 59% editing efficiency of the endogenous target RNA.
finally, the functionality of the precise RNA editing machine achieved by the study was further reflected in the repair and operation of the cracking yeast reverse transcription transmuting Tf1 mutant strain.
Tf1 is similar to the retrovirus in animal cells, and its jumping and amplification require scanrecording of intermediate RNA.
researchers used precision RNA editing to restore the transposing capacity of Tf1 mutant strains, an application that offers a potential tool for retrovirus intervention and operation.
the study was mainly done by Jing Xinyun and Ice Ran et al.
related work has been supported by the Ministry of Agriculture of China, the National Science and Technology Major Project and the Natural Science Foundation project.
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