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

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.

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

“…AAV plasmids were generated via restriction enzyme cloning, all other constructs were cloned by Golden Gate assembly (50). The vector for co-expression of NmeCas9 and the VEGFA sgRNA was previously published by us (51). All other sgRNAs were cloned into plasmid pEJS654 All-in-One AAV-sgRNA-hNmeCas9 (kind gift from Erik Sontheimer, Addgene plasmid #112139) via the SapI restriction sites.…”

“…The dual luciferase reporter was previously reported by us (49). AcrIIC3-LOV2 hybrid constructs were created by inserting the LOV2 domain into our published CMV-driven AcrIIC3 expression vector (Addgene plasmid #120301) (51). To this end, the AcrIIC3 vector was linearized by an around-the-horn PCR using primers carrying BbsI restriction sites as 5' extension.…”

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

“…Moreover, Acrs can be harnessed to enable the temperatureresponsive [41] and optogenetic [42] control of CRISPR-Cas activity. Importantly, Acrs can enhance the editing [38] and cell-type [43] specificities and reduce the cytotoxicity [11] of CRISPR-Cas-mediated genome editing. It has also been reported that Acrs can facilitate the production of CRISPR-carrying viral vectors by restricting CRISPR selfcleavage [44].…”

Anti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering

“…For instance, confining CRISPR technology-based perturbations to selected cell types in genetic studies in animals is critical, and ensuring specificity and hence safety in therapeutic genome editing in human patients is even more critical; thus, Acrs provide a new option to overcome these challenges based on Cas protein function. Hoffmann et al [106] developed a novel cell-type-specific Cas-ON switch based on AcrIIA4 and controlled by cellular miRNA signatures, confining spCas9 activity to hepatocytes or cardiomyocytes. In parallel with that work, Hirosawa et al constructed a synthetic mRNAdelivered Cas9-ON system using miRNA-responsive AcrIIA4 mRNA on the basis of improving the RNAbinding protein L7Ae-based miR-Cas9-ON switch they built previously [107,108].…”

“…In parallel with that work, Hirosawa et al constructed a synthetic mRNAdelivered Cas9-ON system using miRNA-responsive AcrIIA4 mRNA on the basis of improving the RNAbinding protein L7Ae-based miR-Cas9-ON switch they built previously [107,108]. Compared with a Cas-ON switch based on an L7Ae repressor, this Cas-ON switch design based on Acrs showed greater control than the previous system due to the high affinity of Acrs for Cas9-sgRNA complexes [106,107]. Furthermore, a subsequent study validated the efficiency of this Cas-ON switch based on Acrs by adeno-associated virus vectors delivered to adult mice, and the data showed that NmeCas9, along with an AcrIIC3 constructed that was targeted for repression by liver-specific miR-122, allowed editing in the liver while repressing editing in heart muscle, indicating that this strategy provides safeguards against off-tissue genome editing [109].…”