Activation of endogenous full-length active LINE-1 RNA using CRISPR activation to study its role during somatic cell reprogramming

Alsolami, Amjad

Activation of endogenous full-length active LINE-1 RNA using CRISPR activation to study its role during somatic cell reprogramming

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Embargo End Date

2022-11-25

Type

Thesis

Date

2021-11

Access Restrictions

At the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis will become available to the public after the expiration of the embargo on 2022-11-25.

Abstract

The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

Citation

Alsolami, A. (2021). Activation of endogenous full-length active LINE-1 RNA using CRISPR activation to study its role during somatic cell reprogramming. KAUST Research Repository. https://doi.org/10.25781/KAUST-31L5B