Browsing by Subject "scRNA-seq"

A novel method of somatic cell reprogramming employing CRISPR/Cas mediated gene activation (CRISPRa) may lead to improvements in the quality and efficiency of induced pluripotent stem cell (iPSC) generation by directly activating the endogenous factors of the cell. However, this method is yet to be optimized and is inefficient in its current form. Thorough characterization of the molecular events that occur during CRISPRa-mediated reprogramming could permit the fine-tuning of this method to improve iPSC production. B-lymphoblastoid cell lines (LCLs) adhere to culture plates during reprogramming, offering a form of selection for reprogramming cell populations. This thesis aimed to establish a system using LCLs for the characterisation of CRISPRa-mediated pluripotent reprogramming at the single-cell transcriptomic level. In this thesis LCL reprogramming conditions were characterized using alkaline phosphatase staining, immunocytochemistry, embryoid body formation, and live cell imaging. CRISPRa-mediated reprogramming efficiency was greatly increased by the targeting of the miR-302/367 cluster, a group of microRNAs known to improve the efficiency of transgenic reprogramming. Samples were collected for single-cell RNA sequencing (scRNA-seq) at multiple stages of reprogramming, the pluripotency of the iPSC samples was assessed, and a subset of the samples was sequenced. Clustering analysis of the sequencing data showed that the samples clustered apart from one another distinctly based on gene expression. The expression of notable genetic markers of LCLs, pluripotency, and developmental stages was consistent with the loss of somatic cell identity and rise of subpopulations characteristic of reprogramming. These results show that this is a functional system for scRNA-seq sample preparation that can be used to investigate reprogramming kinetics, and the samples collected will be part of a larger study of CRISPRa reprogramming.