Applications of the CRISPR/Cas system beyond gene editing

Abstract

Since the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) as a tool for gene editing a plethora of locus-specific as well as genome-wide approaches have been developed that allow efficient and reproducible manipulation of genomic sequences. However, the seemingly unbound potential of CRISPR/Cas does not stop with its utilization as a site-directed nuclease. Mutations in its catalytic centers render Cas9 (dCas9) a universal recruitment platform that can be utilized to control transcription, visualize DNA sequences, investigate in situ proteome compositions and manipulate epigenetic modifications at user-defined genomic loci. In this review, we give a comprehensive introduction and overview of the development, improvement and application of recent dCas9-based approaches.

Keywords: CASPEX; CRISPR imaging; CRISPR/Cas; CRISPRa; CRISPRi; CasID; dCas9.

Figure 1:

Overview of dCas9-based applications to study and manipulate chromatin. (A) Catalytically inactive Cas9 (dCas9) represents a RNA-guided DNA-binding platform that can be harnessed to target FP to a pre-defined genomic sequence and allows to visualize spatiotemporal dynamics of chromatin in living cells. (B) Fused to a variety of effector proteins, dCas9 can be employed to directly alter the transcriptional state of specific genes or to precisely manipulate epigenetic marks, such as CpG-methylation or post-translational modifications of histones. (C) Additionally, dCas9 can be fused to biotin ligases (tag = BirA* or APEX2). This approach allows to biotinylate (red pentagons) locus-associated proteins and to subsequently identify them by mass spectrometry.

Figure 2:

Expanding the CRISPR imaging toolkit. (A, B) To enhance the signal-to-noise ratio of CRISPR imaging, dCas9 is fused to arrays of small peptide epitopes (GCN4 and sfGFP11). These epitopes then either recruit fluorescent molecules (scFv-GFP) or are complemented (sfGFP1–10), reconstituting fluorescence. (C–E) Multi-color CRISPR imaging can be achieved by either co-expressing differentially labeled orthogonal dCas9 proteins (Sp-dCas9-GFP and Nm-dCas9-RFP) or by fusion of RNA aptamers (PP7 or MS2) to the sgRNA and co-expressing the cognate, fluorescently tagged binding proteins (PCP-GFP and MCP-GFP, respectively). Moreover, sgRNAs can be appended by PUF-binding sites (PBS1 or PBS2, respectively). These sites are then recognized by differentially tagged PUF proteins (PUF1-GFP and PUF2-RFP). (F) By substituting the PAM sequence in the form of an oligonucleotide (PAMmer), dCas9 can be targeted to single stranded RNA molecules.

Figure 3:

Identification of locus associated proteins by dCas9. (A) For enChIP, dCas9 is fused to a FLAG-tag and targeted to a locus of interest. Chromatin is then crosslinked and fragmented. dCas9-bound chromatin fragments are subsequently isolated by FLAG-specific antibodies and analyzed via mass spectrometry. (B) Contrary to enChIP, CasID requires the expression of dCas9 fused to the promiscuous biotin ligase BirA*. After the culture medium has been supplemented with exogenous biotin, BirA* catalyzes the addition of biotin to lysine residues of proteins that are in close proximity to the dCas9-BirA* fusion protein. Lysis of the cells and denaturation of proteins is then followed by affinity purification of biotinylated peptides, which are identified via tandem MS.