UMMS Affiliation

RNA Therapeutics Institute; Program in Molecular, Cell and Cancer Biology; Program in Molecular Medicine

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Document Type



Amino Acids, Peptides, and Proteins | Biochemistry | Genetics and Genomics | Hemic and Immune Systems | Immunity | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides | Tissues


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 editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). 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 tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural 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 anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3 Nme construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.


AAV, Cas9, anti-CRISPR, microRNA, tissue-specific editing

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© 2019 Lee et al. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at

DOI of Published Version



Lee J, Mou H, Ibraheim R, Liang SQ, Liu P, Xue W, Sontheimer EJ. Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins. RNA. 2019 Nov;25(11):1421-1431. doi: 10.1261/rna.071704.119. Epub 2019 Aug 22. PMID: 31439808; PMCID: PMC6795140. Link to article on publisher's site

Journal/Book/Conference Title

RNA (New York, N.Y.)

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Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

Available for download on Sunday, November 01, 2020