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

Determining the recruitment relationships of nuclear proteins is essential for understanding the mechanisms underlying nuclear complex assembly and gene regulation. A widely used method for studying recruitment is chromatin immunoprecipitation (ChIP), but it requires fixation, chromatin shearing, and specific antibodies and cannot easily resolve recruitment directionality. Other systems like lacO/LacI are restricted to a limited number of specialized cell lines containing this lacO array’s integration. To overcome these limitations, we developed a novel microscopy-based assay, CRISPR-PITA (protein interaction and telomere recruitment assay), to assess whether a nuclear protein can recruit other nuclear factors in living cells. The protein of interest is targeted to repetitive genomic loci (e.g., telomeres) using catalytically inactive Cas9 (dCas9) fused to a SunTag array, resulting in visible nuclear foci. Recruitment of endogenous proteins is evaluated by immunofluorescence. For proof-of-concept, we tested the Kaposi’s sarcoma herpesvirus (KSHV) latency-associated nuclear antigen (LANA). CRISPR-PITA revealed that LANA recruits known interactors, such as ORC2 and SIN3A, but not MeCP2. Conversely, MeCP2 recruits LANA, indicating a unidirectional recruitment relationship. Similarly, MeCP2 could recruit HDAC1, while HDAC1 could not recruit MeCP2, further supporting directional nuclear interactions. Here, we present an easy, straightforward protocol applicable to any transfectable cell line, enabling researchers to dissect recruitment dynamics at high spatial resolution. CRISPR-PITA provides a powerful, flexible, and accessible platform to interrogate recruitment directionality between nuclear proteins in their native cellular context.