Hostname: page-component-84b7d79bbc-g78kv Total loading time: 0 Render date: 2024-07-28T02:28:54.370Z Has data issue: false hasContentIssue false
  • English
  • Français

Development of a self-replicating plasmid for non-toxic expression of CRISPR-repressors to study schizophrenia-risk genes

Published online by Cambridge University Press:  01 September 2022

D. Abashkin ,
A. Kurishev ,
D. Karpov  and
V. Golimbet
D. Abashkin
Affiliation:
Mental Health Research Center, Clinical Genetics Laboratory, Moscow, Russian Federation
A. Kurishev
Affiliation:
Mental Health Research Center, Clinical Genetics Laboratory, Moscow, Russian Federation
D. Karpov
Affiliation:
Mental Health Research Center, Clinical Genetics Laboratory, Moscow, Russian Federation
V. Golimbet*
Affiliation:
Mental Health Research Center, Clinical Genetics Laboratory, Moscow, Russian Federation
*
*Corresponding author.

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.
Introduction

Genome-wide association studies revealed that polymorphisms located within non-coding regions significantly contribute to the genetic architecture of schizophrenia. Such regions may affect the expression of tens and hundreds of neuronal genes. Epigenetic CRISPR editors help to elucidate the causative polymorphisms. However, efficient CRISPR-repressors are highly toxic to neuronal cells, and their activity rapidly declines with time after transfection due to plasmid silencing. Therefore, less toxic, effective, and long-acting epigenetic CRISPR instruments are required to advance schizophrenia genetic research.

Objectives

We aimed at creating a less toxic and effective CRISPR-repressor for the investigation of schizophrenia-risk genes.

Methods

Plasmids were obtained used standard molecular cloning techniques and lipofected into the SH-SY5Y cell line. Cells were cultured using standard conditions and techniques. Cell viability and GFP-reporter fluorescence were observed using a fluorescent microscope.

Results

We obtained a set of plasmids encoding dCas9-KRAB-MeCP2 repressor under the control of different promoters (hEF1a, hPGK1, mPGK1, hSYN2, synthetic TRE). Non-toxic expression of the CRISPR-repressor was achieved using tetracyclin controllable TRE promoter. Moreover, the Epstein-Barr virus origin of replication (oriP) and its regulator EBNA were introduced to make the self-replicating plasmid. High activity of CRISPR-repressor was confirmed on a schizophrenia-risk gene DDC encoding L-DOPA decarboxylase catalyzing the last step of dopamine biosynthesis.

Conclusions

We have created a plasmid encoding the non-toxic and effective CRISPR repressor encoded by a self-replicating plasmid. The study was supported by the grant from the Russian Science Foundation №21-15-00124, https://rscf.ru/project/21-15-00124/.

Disclosure

No significant relationships.

Keywords

causative genesschizophréniaCRISPR editors
Type
Abstract
Information
European Psychiatry , Volume 65 , Special Issue S1: Abstracts of the 30th European Congress of Psychiatry , June 2022 , pp. S152
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
© The Author(s), 2022. Published by Cambridge University Press on behalf of the European Psychiatric Association
Submit a response

Comments

No Comments have been published for this article.