Unidirectional recruitment relations between proteins can be determined by a CRISPR/dCas9 recruitment assay

Directional recruitment of protein complexes is critical for proper function of many nuclear processes. Here we present CRISPR-PITA (Protein Interaction and Telomere Recruitment Assay), an assay that determines the ability of a given protein to recruit any other nuclear factor. The protein of interest is directed via CRISPR/dCas9, a dead Cas9 that does not cut DNA, to a repeat sequence, such as telomeres, to obtain dots that are easily detectable by microscopy. The recruitment of endogenous nuclear proteins to these dots can then be visualized using specific antibodies. We determined recruitment abilities in CRISPR-PITA to methyl-CpG binding protein MeCP2, histone deacetylase 1 (HDAC1), heterochromatin protein 1 (HP1α), and the latency-associated nuclear antigen (LANA) encoded by Kaposi’s sarcoma associated herpesvirus (KSHV, HHV-8). LANA was able to recruit its known interactors ORC2 and SIN3A to LANA-telomere dots. In contrast, LANA was unable to recruit MeCP2 whereas MeCP2 was able to recruit LANA. Similarly, HDAC1 that interacts with MeCP2 through the transcriptional-repression domain (TRD) same as LANA, was unable to recruit MeCP2, but MeCP2 recruited HDAC1. One important function of LANA is to tether the viral episomal genomes to the cellular chromosomes during cell division. The unidirectional recruitment of LANA by MeCP2, makes MeCP2 a candidate anchor for KSHV genome tethering by LANA. We found that cells derived from Rett syndrome and express a mutant MeCP2 (T158M), impaired in DNA binding, cannot support KSHV genome maintenance. In summary, we describe a broadly applicable protein recruitment assay based on CRISPR/dCas9.Significance StatementCRISPR/Cas9 is a revolutionary system that has profoundly impacted biology research. Here we present another application for CRISPR/Cas9, in evaluating recruitment relations between nuclear proteins. A protein of interest is directed to a repeat sequence via the catalytically inactive Cas9 (dCas9) to generate easily detectable dots. Then, the recruitment of other nuclear proteins to these dots can be evaluated. Using this assay, we show that some interacting proteins have a unidirectional recruitment property, where only one of the proteins can recruit its partner. We propose that available interacting domains can force this unidirectional recruitment. Using this recruitment assay, we found unidirectional recruitment of the KSHV encoded LANA and HDAC1 by MeCP2. Furthermore, this unidirectional recruitment is critical for viral latency, since LANA fails to maintain the viral genomes in MeCP2 mutant cells.