Generation of CRISPR-cas9 construct for knockout of genes encoding chromatin-associated proteins

Abstract

Background: Eukaryotic genomes have a multiscale three-dimensional organization varying from nucleosomes, loops, topologically associating domains, and chromosome territories. Chromatin, DNA wrapped around histone proteins, helps in packaging long DNA within tiny nuclear spaces. We used CRISPR-cas9, which is a system of single-protein and single-guide RNAs for genome engineering and also is simple and target specific.Method: Two major protein families involved in maintaining and regulating structure and dynamics of chromatin are trithorax group (TrxG) and polycomb group (PcG) proteins. This study was undertaken to generate knockout cell lines of some TrxG and PcG proteins using the CRISPR-based approach in order to study their role in higher order chromatin organization. Results: From TrxG, ISWI and Acf were selected, and from PcG, Pc and Psc were selected. Three pAc-sgRNA-Cas9puro-vector constructs for ISWI gene, one pAc-sgRNA-cas9-puro-vector construct for Pc, gene and two pAc-sgRNA-cas9-puro-vector constructs for each of the Acf and Psc gene were generated. These constructs were confirmed by PCR and sequencing.Conclusion: In the future, these constructs will be used to study the role of their respective target genes in chromatin organization.