Generation of cell lines with homozygous deletions using CRISPR-CAS to study the effect of linear distance on enhancer function

Abstract

Enhancers are cis-regulatory elements that affect the transcription rate of a subset of genes by interacting with their respective promoter. During the last few years, it has been found that these enhancers regulate genes found in the same topologically associated domain (TAD), a region delimited by boundary conditions in form of CTCF loops for the loop extrusion activity of cohesin. This way enhancers only affect genes found in the same TAD. However, these genes are not regulated equally within the TAD, and gene expression vary depending on several factors, like the promoter-enhancer distance or the regions close to them like the presence of tethering elements as CpG islands, present in various degree determined by the type of gene, if it is a housekeeping, developmental or tissue-specific. Therefore, in this study we aim to generate cell lines with homozygous clones for a deletion achieved by novel geneediting technique CRISPR-CAS, as a way to bring closer the promoter of Rp1l1, a tissuespecific gene, to a rich-CGI enhancer (poised enhancer) situated between Rp1l1 and SOX7, a developmental gene within the same TAD. We aim generate at least two clones for two different cell lines, a wildtype cell line without the enhancer (WT) and another cell line with the poised enhancer (PE). Future studies will cover the effects of distance on gene expression of both Rp1l1 and SOX7, considering the presence or absence of CpG islands coupled to the enhancer and how it affects the promoter-enhancer communication.