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

Hoffmann, M. D.; Aschenbrenner, S.; Grosse, S.; Rapti, K.; Domenger, C.; Fakhiri, J.; Mastel, M.; Eils, R.; Grimm, D.; Niopek, D.

doi:10.1101/480384

bioRxiv · 10.1101/480384

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

Hoffmann, M. D.·Aschenbrenner, S.·Grosse, S.·Rapti, K.·Domenger, C.·Fakhiri, J.·Mastel, M.·Eils, R.·Grimm, D.·Niopek, D.

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Abstract

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 muscle cells, respectively, into the 3UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, 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 activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.

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Hoffmann, M. D., Aschenbrenner, S., Grosse, S., Rapti, K., Domenger, C., Fakhiri, J., Mastel, M., Eils, R., Grimm, D., Niopek, D.. 2018-11-27. Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins. https://doi.org/10.1101/480384

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