CRISPR-Cas9 induced knockout for the functional study of Myef2’s role in X-chromosome inactivation

Abstract

Across animals of all kinds there are many different forms of dosage compensation in order to equalize gene expression between the sexes. One widely known form is known as X chromosome inactivation (XCI). In this process females compensate for their extra X chromosome by randomly choosing one to be inactivated, returning to a level of X chromosome gene expression comparable to males. Although XCI has been studied and looked into since as early as the 1960s, certain intricacies of its mechanism still elude us. This study aimed to utilize a CRISPR/Cas9 system for the purpose of targeting and knocking out Myef2 in a mouse embryonic stem (mES) cell line. Our choice mES cell line (AV3.1.12) is an XY cell line containing a doxycycline-inducible Xist knock in. A knockout (KO) cell line allows for future studies and also to preliminarily determine its potential role and significance in XCI. In our study we created and tested the knockout cell line by inducing cell death by Xist-induction and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Our results showed that against our expectations, in the KO cell line, cell survival post-XCI was decreased significantly while Myef2 transcript experienced a 3.36 fold decrease in expression.