Development of a tool to introduce DNA methylation into a genomic locus in vivo

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Gaurav Phuyal (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Ramji Bhandari

Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (Cas9) system, also called CRISPR-Cas9, has been manipulated for application in a wide variety of biomedical research. The simplicity of the CRISPR system has made it easy for its successful application in introducing both genetic and epigenetic modifications in vitro and in vivo. While essential for development and differentiation in organisms, epigenetic processes (DNA Methylation, Histone modification, and non-coding RNAs) have been found to be influenced by endogenous, biotic, and abiotic factors. Epigenetic alterations, particularly DNA methylation changes that occur due to early-life environmental exposures have been identified as one of the potential mechanisms underlying the developmental origins of health and disease (DOHaD). The Cas9 protein can be made devoid of its endonuclease activity resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. Transcriptional effectors are proteins that can activate or repress gene transcription. Although a few experiments have been performed using transcriptional effectors in the Crispr-dcas9 system in vitro, an effective and reproducible technique to epigenetically activate/suppress genes using activator and repressor elements in vivo has not been developed. Given that embryos undergo epigenetic reprogramming of parental epigenetic marks during the cleavage stages, it is difficult to determine if epigenome targeting at the one-cell stage can escape genome-wide reprogramming of the embryo occurring at cleavage stages. It is also unknown if de novo-established epigenetic alterations are programmed differently from those inherited from parental gametes. To date, there have been no studies demonstrating the fate of epigenetic edits during epigenetic reprogramming in vivo. This study examines if the epigenome editing performed at zygotic stages will alter expression of a heritable epigenetic trait that can withstand the reprogramming window during the subsequent cleavage stages. To this end, we are using a fish model medaka (Oryzias latipes) which has been established as an excellent biomedical model given its similarity in germ cell development to mouse and humans and applying CRISPR-dCcas9-DNMT3aa repressor at the first few cell stages. This study will help delineate the applicability of the CRISPR system in introducing epigenetic modifications during the epigenetic reprogramming windows.

Additional Information

Language: English
Date: 2023
CRISPRdCas9, Epigenetics, Medaka, Transgenerational inheritance
CRISPR-associated protein 9
DNA ‡x Methylation
Oryzias latipes

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