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

Mareike D Hoffmann,Claire Domenger,Manuel Mastel,Roland Eils,Dominik Niopek,Kleopatra Rapti,Stefanie Grosse,Kathleen Börner,Julia Fakhiri,Sabine Aschenbrenner,Dirk Grimm

doi:10.1093/nar/gkz271

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 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.

Highlights

CRISPR technologies provide an efficient and simple means to perform targeted genetic manipulations in living cells and animals [1,2,3,4,5]
To generate a cell-specific Cas9-ON switch, we aimed at rendering the activity of CRISPR–Cas9 dependent on the presence of cell-specific miRNAs, i.e. miRNAs that are abundant solely within the target cell type
Anti-CRISPR proteins block CRISPR–Cas9 DNA targeting, Cas9 nuclease function or both by directly binding to the Cas9/sgRNA complex. This post-translational inhibitory mechanism enables a complete shutdown of CRISPR–Cas9 activity even upon simultaneous delivery of Cas9, a sgRNA, and an antiCRISPR-encoding vector [29,30,31,44]

Summary

Introduction

CRISPR (clustered regularly-interspaced short palindromic repeats) technologies provide an efficient and simple means to perform targeted genetic manipulations in living cells and animals [1,2,3,4,5]. The rapidly expanding CRISPR toolbox enables genomic knock-ins/-outs [2,3], gene silencing and activation [6,7,8], epigenetic reprogramming [9,10,11], as well as single-base editing [12,13]. These tools facilitate detailed genetic studies in cells and animals and hold enormous potential for the future treatment of genetic disorders [14]. Virtually any mode of efficient in vivo delivery of the CRISPR–Cas components (e.g. via viral vectors, nanoparticles, lipophilic complexes etc.) is likely to affect many cell types and tissues beyond the one of ac-

Methods

Results

Conclusion