Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed...

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Bibliographic Details
Published in:	Nucleic acids research 2019-07, Vol.47 (13), p.e75-e75
Main Authors:	Hoffmann, Mareike D, Aschenbrenner, Sabine, Grosse, Stefanie, Rapti, Kleopatra, Domenger, Claire, Fakhiri, Julia, Mastel, Manuel, Börner, Kathleen, Eils, Roland, Grimm, Dirk, Niopek, Dominik
Format:	Article
Language:	eng
Subjects:	3' Untranslated Regions - genetics Binding Sites CRISPR-Associated Protein 9 - antagonists & inhibitors CRISPR-Associated Protein 9 - biosynthesis CRISPR-Associated Protein 9 - genetics CRISPR-Cas Systems Dependovirus - genetics Enzyme Activation Enzyme Induction Gene Editing - methods Gene Expression Regulation Genes, Reporter HEK293 Cells HeLa Cells Hepatocytes - metabolism Humans Luciferases, Renilla - analysis Luciferases, Renilla - genetics Methods Online MicroRNAs Myocytes, Cardiac - metabolism Organ Specificity Protein Isoforms - antagonists & inhibitors Transgenes
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Summary:	The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.
ISSN:	0305-1048 1362-4962