CRISPR-PITA: An Imaging-Based CRISPR/dCas9 Assay to Determine Recruitment Directionality of Nuclear Proteins

Background

The study of protein–protein and protein–chromatin interactions is fundamental for understanding cellular regulation and nuclear organization. Widely used biochemical approaches, such as co-immunoprecipitation (co-IP), enable the detection of protein complexes by selectively enriching interacting partners via a specific antibody. However, while co-IP is effective for identifying protein interactions, it does not provide spatial or hierarchical information and cannot determine the directionality of recruitment, i.e., which protein acts as the primary recruiter and which proteins are subsequently assembled.

Understanding protein recruitment to specific genomic regions is therefore essential for dissecting mechanisms of chromatin organization and transcriptional regulation. Several assays have been developed to investigate protein–DNA or protein–chromatin interactions, with chromatin immunoprecipitation (ChIP) being one of the most widely used methods [1]. ChIP allows the detection of proteins bound to defined genomic loci, and, in some cases, can provide indirect information on chromatin-associated protein complexes, for example, through ChIP-based proteomic approaches that identify proteins co-associated with specific loci [2]. However, the technique requires fixation, chromatin fragmentation, and immunoprecipitation with high-quality antibodies that can recognize cross-linked proteins. In addition, when multiple proteins are found at the same genomic site, ChIP alone does not resolve the order or directionality of recruitment [3].

To overcome this, ChIP is sometimes combined with genetic perturbations such as knockout (KO) [4] or knockdown (KD) [5] of a suspected recruiter protein. However, these approaches are time-consuming and require extensive validation. An alternative approach relies on the integration of the bacterial lac operator (lacO) array into the host genome and tethering of a protein of interest via fusion to the Lac repressor (LacI) [6]. This system enables recruitment of candidate proteins to a defined genomic locus and allows visualization by microscopy. However, its applicability is limited by the availability of suitable cell lines carrying lacO arrays, and it does not readily support reciprocal or bidirectional recruitment analyses. To address these limitations, we developed a simple, flexible method to test protein recruitment relations in living cells [7]. This approach utilizes dCas9 targeted to repetitive genomic sequences (telomeres) in combination with the SunTag system [8], which amplifies the signal by generating robust, microscopically visible nuclear foci. Briefly, the SunTag system consists of a tandem array of peptide epitopes fused to dCas9, which recruits multiple copies of a cognate single-chain antibody (scFv) fused to the protein of interest, thereby amplifying the local signal at the targeted locus and facilitating the detection of potential interactions. Proteins recruited to these foci can be detected by using antibodies that recognize native epitopes or by tagging the proteins with fluorescent markers (Figure 1). While targeting dCas9 to telomeric repeats has previously been employed to study nuclear dynamics, including heterochromatin organization and liquid–liquid phase separation (LLPS), it has not been adapted to systematically investigate recruitment directionality [9,10].

Our protocol does not require chromatin cross-linking or shearing and does not depend on specialized cell lines or complex genetic manipulations. It allows flexible testing of both direct and reciprocal recruitment relationships, making it broadly applicable to studies of chromatin-associated factors, transcriptional complexes, and virus–host interactions. Using this approach, we have successfully identified unidirectional recruitment dependencies between nuclear proteins, demonstrating the method’s value in dissecting functional hierarchies within protein complexes.

Figure 1. Recruitment phenotypes detected using the CRISPR-PITA assay. (A) Negative recruitment: The recruiter protein (“protein Y,” red) is enriched at repetitive telomeric regions, while the protein of interest (“protein X,” green) remains diffusely distributed throughout the nucleus, indicating the absence of recruitment (left). Positive recruitment: Both the recruiter protein and the protein of interest co-localize at repetitive telomeric regions, resulting in a merged yellow signal, indicative of positive recruitment (right). (B) The recruiter protein LANA is enriched at repetitive telomeric regions (red), while the protein of interest MeCP2 (green) remains diffusely distributed throughout the nucleus, indicating the absence of recruitment. (C) Both the recruiter protein LANA (red) and the protein of interest ORC2 (green) co-localize at repetitive telomeric regions, resulting in a merged yellow signal, indicative of positive recruitment. HCT cells (panel B) and SLK cells (panel C) were transfected with CRISPR-PITA components, as can be seen in the illustrations on the left of each panel. Upper panel: transfection with dCas9-SunTag, Fc-LANA, and sgRNA. Middle panel: transfection with Fc-LANA and sgRNA. Lower panel: transfection with dCas9-SunTag and sgRNA. Immunofluorescence assays were performed to detect mouse anti-LANA antibody (red, Alexa Fluor 594) and rabbit anti-MeCP2 or rabbit anti-ORC2 (green, Alexa Fluor 488) cellular localization. The nucleus was stained with DAPI. Scale bars = 5 μm.