Dead Cas Systems: Types, Principles, and Applications

Abstract

The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.

Keywords: Cas9; cancer; chromatin; dCas; editing; epigenetics; hereditary diseases; infectious diseases; inflammatory diseases; transcription.

Figure 1

Types and applications of dCas-based molecular tools. (A) Investigation of chromatin structure. dCas proteins tethered with specific enzymes (e.g., peroxidase) enable inducible marking (biotinylation) of chromatin factors in the vicinity of the target site. These factors can be subsequently analyzed by proteomics to study chromatin organization. (B) Base editing. dCas proteins coupled with base editing enzymes (cytidine or adenine deaminases) can be used to modify RNA or DNA, correct genetic mutations, or knock-out genes. (C) Epigenetic remodeling. dCas-based epigenome modifiers can directly alter epigenetic state at a given locus, which is frequently used to annotate gene regulatory elements. Red and green spheres indicate heterochromatin and euchromatin marks, correspondingly. (D) Programming 3D chromatin interactions. Using two dCas proteins targeting defined genomic loci can program 3D chromatin interactions. A chemical inducer stimulates dimerization of dCas proteins fused with dimerization domains building long-range connections between genomic elements. (E) Transcriptional regulation. Control of gene expression by dCas proteins tethered to transcriptional suppressors (red) or activators (green). PAM—protospacer adjacent motif; H840A and D10A are point mutations inactivating catalytic residues RuvC and HNH, correspondingly. This picture was created in BioRender software.

Figure 2

Modification of CRISPR components for improved epigenetic regulation. (A) SunTag technique. dCas is fused with GCN4 peptide array, which attracts any effector molecule containing single-chain variable fragments (scFV). Multiple GCN4-scFV interactions ensure efficient recruitment of many effector molecules to the dCas-programmed genomic site. (B) Scaffold technique. In Scaffold, effector molecules are recruited to the target site via the interaction of MCP aptamer-specific protein with a short synthetic gRNA containing MS2 aptamer. gRNA protrudes out of the Cas-gRNA complex, so that chimeric gRNA-MS2 transcripts efficiently recruit effectors carrying MCP molecules. (C) TREE combines SunTag and Scaffold techniques, providing additional recruitment of effector molecules. (D) SAM is based on a two-component transcriptional effector (p65-HSF1) recruited to the target via MS2-MCP interaction. Additionally, dCas protein is tethered to a transcriptional regulator (VP64) to increase potency of the effect. This picture was created in BioRender software.