RNA Therapeutics Institute; Program in Molecular Medicine; Graduate School of Biomedical Sciences
Amino Acids, Peptides, and Proteins | Bioinformatics | Computational Biology | Molecular, Cellular, and Tissue Engineering | Nucleic Acids, Nucleotides, and Nucleosides
CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from persistent activity of Cas9 after the desired editing is complete, or from editing activity in unintended cell types or tissues upon in vivo delivery [e.g. by adeno-associated viruses (AAV)]. Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropisms are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs), which are proteins evolved as countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3’ UTR, and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective Acrs. Furthermore, we demonstrate that in vivo delivery of Cas9 and Acrs that are targeted for repression by liver-specific miR-122 allow editing in the liver while Acrs devoid of miRNA regulation prevent Cas9 activity. This strategy provides additional safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.
bioengineering, Cas9, anti-CRISPR proteins, genome editing
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
DOI of Published Version
bioRxiv 631689; doi: https://doi.org/10.1101/631689. Link to preprint on bioRxiv service.
Lee J, Mou H, Ibraheim R, Liang S, Xue W, Sontheimer EJ. (2019). Tissue-specific Genome Editing in vivo by MicroRNA-repressible Anti-CRISPR Proteins [preprint]. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/631689. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1687
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