Non-targeting guide is the same as in Fig. D) PspCas13b and LwaCas13a knockdown activity (as measured by luciferase activity) using tiling guides against Cluc. C) PspCas13b and LwaCas13a knockdown activity (as measured by luciferase activity) using tiling guides against Gluc. coli LacZ transcript, with no homology to the human transcriptome. Values are normalized to a non-targeting guide with designed against the E. Orthologs with efficient knockdown using both guides are labeled with their host organism name. B) Evaluation of 19 Cas13a, 15 Cas13b, and 7 Cas13c orthologs for luciferase knockdown using two different guides. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.Ĭopyright © 2017, American Association for the Advancement of Science.Ī) Schematic of stereotypical Cas13 loci and corresponding crRNA structure. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products.
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