Özet:
Genomic DNA is maintained and regulated by chromatin proteins. Methods for research of chromatin organization are numerous and established, however, it is still a challenge to identify novel chromatin proteins associated with a speci c locus. In the scope of this project, we work on implementing a novel tool for the identi cation of chromatin proteins associated with a given genomic region of interest. The method we used is based on combining the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and SICAP (Selective Isolation of Chromatin-Associated Proteins) techniques. CRISPR technique is used for targeting the aimed locus, and SICAP technique provides bene ts by employing two tandem pulldowns. First pulldown is dCas9-mediated and locus-speci c, while second is based on streptavidin binding to in vitro-biotinylated DNA of chromatin fragments. Second pulldown enables to get rid of contaminants by stringent denaturing washes and limits contaminant proteins to the chromatin. Resulted pool of chromatin proteins is eluted, and is ultimately aimed to be analyzed with mass spectrometry. In current work, most of the critical components of the method were developed and veri ed. Also, we implemented the model genomic locus for the validation of the method. CRISPR-SICAP method was then probed on di erent genomic regions by CRISPR-SICAP-qPCR and was shown to be more speci c than regular dCas9- ChIP. These results can be extrapolated to future applications to locus-speci c protein research. Both CRISPR-SICAP and model genomic loci are optimized and ready for being applied for obtaining pool of locus-associated chromatin proteins.
