CRISPR-PITA: A NOVEL CRISPR/dCas9 BASED ASSAY TO DETERMINE RECRUITMENT RELATIONS BETWEEN PROTEINS

We present a novel assay named CRISPR-PITA (Protein Interaction and Telomere recruitment Assay), to determine the ability of any nuclear protein to recruit any other nuclear factor in protein interaction studies. We target the protein of interest to a repeated sequence, such as telomeres, in order to obtain easily detectable dots in microscope following immunostaining. Then using specific antibodies against endogenous nuclear proteins, we ask if a second protein was recruited to the telomeric dots. For a proof of concept, we used the latency-associated nuclear antigen (LANA) encoded by Kaposi’s sarcoma associated herpesvirus (KSHV, HHV-8). In infected cells LANA is localized to the viral episomal genomes, known as LANA dots, but without the viral genomes LANA is equally distributed in the nucleus. This makes it hard to determine if LANA or the viral genomes are responsible for the recruitment of various nuclear proteins that were detected as LANA interacting proteins in biochemical assays. For that purpose, we utilized the CRISPR/dCas9, a dead Cas9 that does not cut DNA, and LANA was targeted to this dCas9 via the Sun-tag system. Using this CRISPR-PITA, we observed that some LANA interacting proteins were recruited by LANA to the telomeres, but some proteins that were previously detected with LANA dots were not recruited by LANA to the telomeres. Interestingly, we find that the recruitment does not always work bi-directionally. In summary, we describe a simple recruitment assay based on CRISPR/dCas9.