A switchable anti-CRISPR platform for dynamic and dosed CRISPR-Cas genome perturbations

• Applicant: Professor Dr. Dominik Niopek
• Subject Area: General Genetics and Functional Genome Biology ; Structural Biology ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 453202693

The CRISPR-Cas technologies revolutionized research in the life-sciences and hold enormous potential for the treatment of genetic diseases. To advance the accuracy of CRISPR perturbation studies and safeguard the clinical translation of CRISPR (epi)genome editing, the ability to switch ON and OFF or fine-tune the activity of Cas effectors with high precision in time and space is essential. Anti-CRISPR proteins (Acrs) are bacteriophage-derived, potent CRISPR inhibitors that present a novel, largely unexplored regulatory layer of CRISPR systems. In their natural form, Acrs are constitutive inhibitors that suppress Cas activity whenever present in a cell. Here, we propose the creation of a next-generation CRISPR regulatory platform based on switchable Acrs, i.e. engineered, conditional inhibitors that tie Cas activity to the presence or absence of exogenous stimuli. Using a combination of bioinformatics and wet-lab approaches, we will first map allosteric surface sites on a selected panel of Acrs, namely AcrIIA4, AcrIIA5, AcrIIC3, AcrVA1 and AcrVIA5. Together, these Acrs target all Cas9, Cas12 and Cas13a orthologues commonly used today. We will then create hybrids between these Acrs and different sensory domains, thereby rendering Cas inhibition dependent on specific inputs, namely light (blue light, far-red light) or clinically approved drugs (rapamycin, 4-hydroxytamoxifen). The resulting, switchable Acrs will facilitate timely and spatially confined genome editing and transcriptome editing via catalytically active Cas9, -12 and -13. Concurrently, our switchable Acrs will also enable real-time control of d(ead)Cas-effector fusions with customizable functions. To demonstrate the utility of our original regulatory platform, we will employ sets of switchable Acrs to implement logic gates that compute Cas activity in relation to selected input combinations. Finally, we will combine the engineered, light-inducible Acrs with epigenetic CRISPR effectors. This will allow us to study gene expression dynamics in response to spatiotemporally confined epigenetic changes, which is of great interest, for instance, in context of cell differentiation or carcinogenesis. Taken together, our switchable anti-CRISPR toolbox will enable the interrogation of eukaryotic gene and genome regulation, and facilitate novel CRISPR applications in industry and medicine.

• DFG Programme: Research Grants