Edició del genoma de la línia cel·lular HEK293T per gens associats a la Mort Sobtada mitjançant CRISPR/Cas9

Abstract

The CRISPR/Cas9 technique is a gene-editing system recently discovered that can target and modify DNA with great accuracy.This system is based on an endonuclease Cas9 and RNA guides (sgRNAs) which brings the protein to the DNA target location so it cuts the two strands of DNA. The cell will repair this cut introducing specific mutations in the genome. In this project we want to study the efficiency of different RNA guides to edit two genes that may be related to the Sudden Cardiac Death, ZNF394 and SCN10A, in HEK293T cells (kidney cells) genome by CRISPR/Cas9 technique. These guides were previously designed in the Cardiovascular Genetics Centre. It is considered Sudden Cardiac Death (SCD) that natural dead for cardiac causes which occurs quickly and unexpectedly in apparently healthy individuals. It happens during the first hour from the beginning of the symptoms. It is believed that genetic variants of ZNF394 and SNC10A may play some role in the SCD. The main purpose of this work is to discover which sgRNAs combinations will allow a major efficiency in editing by CRISPR/Cas9, before performing in induced pluripotent stem cells (iPSCs). Thus, we cloned the RNA guides into expression vectors, px441 and px462, which express the Cas9 nuclease gene, and we transfected different combinations of them in HEK293T cells, that are an immortalized cellular line easy to transfect and manipulate. Transfection images were obtained and then analyzed. The transfected cells were selected and isolated to extract their DNA that was further sequenced. Analyzing the edition efficiency of each combination we noted that SNC10A gene variant has been introduced in homozygosisin HEK293T cells with some of the sgRNA combinations through CRISPR/Cas9. The best combination found in this study is SCN10A 3 + SCN10A 5 + ssODN sense. It remains the sequencing of ZNF394 gene and it would be necessary to optimize the sequencing of some clones to complete the results