Dmitrii Abashkin, Russian Federation
Mental Health Research Center clinical geneticsAuthor Of 1 Presentation
Epigenome editing of the potential enhancers of the schizophrenia risk genes - EPV0651
Abstract
Introduction
Hereditary factors contribute significantly to the development of schizophrenia. However, the genetic architecture and mechanisms of schizophrenia development are not well understood. Genome-wide analyses of genetic associations in non-coding regions of the genome point out to enhancers as one of the loci associated with an increased risk of schizophrenia.
Objectives
Development of the CRISPR/SpyCas9 repressor system to elucidate the contribution of enhancers in molecular mechanisms associated with the schizophrenia in the model neuronal cell lines.
Methods
A modified chromosome conformation capture Hi-C technique was used to identify enhancer-promoter contacts in neuronal cell lines. Classical molecular cloning was used to construct plasmid and lentiviral vectors bearing CRISPR-repressors and dual-guide RNAs. Lentiviral particles were prepared using HEK293T cell line and the 3d generation package plasmids. SK-N-SH cell line was transfected or infected, followed by selection on puromycin, genome DNA preparation, and estimation of methylation profiles using methylation-sensitive high resolution melting (MS-HRM) analysis.
Results
We identified many neuron-specific enhancers associated with an increased risk of schizophrenia. We have constructed several plasmids and lentiviruses encoding the most robust repressor protein dSpyCas9-KRAB-MeCP2 to target some of these enhancers as well as promoter regions contacted with them. The activity of the repressor is shown, for example, for enhancer of EPHX2 gene.
Conclusions
CRISPR/SpyCas9 repressor system can be used to investigate enhancer-promoter contacts to find out the molecular mechanisms contributing to the development of schizophrenia. The work was supported by the RFBR grant №19-015-00501.