Development & application of novel methods to identify locus-specific transcriptional regulators

Chromatin-associated factors play important roles in the regulation of gene expression. It is essential to identify these factors in order to elucidate their functional roles at the target sites. There are a number of methodologies to identify these chromatin regulators in a locus-specific manner, however, these approaches have their own drawbacks and limitations. In this study, we aim to develop a new method called "Split-CRISPR-ID" to identify locus-specific transcriptional regulators by combining the flexible genomic locus targeting power of CRISPR proteins with in vivo biotinylation-based purification, and proteomic analysis. We set up the system by using two distinct CRISPR proteins fused with the inactive halves of target promiscuous mutant BirA* biotin ligase. We first validated the targeting efficiencies of the CRISPR proteins at various loci on the genome. Then, we created CRISPR-BirA* halves fusion proteins to control the in vivo biotinylation event spatiotemporally target specific loci. Our preliminary data shows heterodimerization and activation of BirA* at designed genomic loci. Further studies will provide the proteomic discovery when the method is fully implemented. In parallel, we also aimed to optimize and apply in our laboratory a recently published method for locus-specific protein discovery called "CAPTURE". CAPTURE is based on the principle of biotinylation and isolation of a nuclease-deficient Cas9 with associated chromatin proteins. Here, we validated the specificity of targeting via various approaches. We envision that the development and application of such improved locus-specific protein identification methods will help us discover novel locus-specific transcriptional regulators, and contribute to the development of novel therapeutic strategies.