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CRISPR/Cas9-mediated interrogation of a sense-antisense pair involved in the DNA damage response

Goyal, Ashish

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Abstract

Previously, the Diederichs lab identified the long non-coding RNA NOP14-AS1 to be induced in A549 and HepG2 cells treated with the DNA-damaging drugs Etoposide, Cisplatin, and Bleomycin. Preliminary data indicated that NOP14-AS1 was inversely co-regulated with its antisense protein-coding gene NOP14. In this study, NOP14-AS1 regulation in DNA damage was further characterized. NOP14-AS1 was consistently induced in several cell lines upon treatment with various DNA damaging agents. NOP14-AS1 induction was a p53-dependent transcriptional response. The DNA damage-induced inverse co-regulation between NOP14 and NOP14-AS1 was further confirmed in multiple cell lines using time-course expression analysis upon treatment with multiple DNA-damaging agents. NOP14 repression upon DNA damage was p53-dependent and preliminary experiments performed indicated its involvement in the p53 pathway and a role in regulating cell proliferation. Antisense lncRNAs often regulate the expression of their overlapping sense protein-coding genes through transcript- as well as transcription-dependent mechanisms. Therefore, it was proposed that NOP14-AS1 induction could lead to NOP14 repression in cis. The transcript-dependent effects of NOP14-AS1 could be uncovered using an RNA interference-/ antisense oligo-based loss-of-function approach. However to uncover any transcription-dependent effects, a CRISPR/Cas9-based knockdown (CRISPRi) and overexpression (CRISPRa) system for NOP14-AS1 was established. While establishing this system, a major limitation of the CRISPR/Cas9 system for lncRNA knockdown was discovered and experimentally demonstrated for multiple examples. CRISPR/Cas9 targeting of a lncRNA arising from a complex locus can unintentionally affect the expression of its neighboring genes advocating for caution while using these systems for knockdown. However, these systems were determined to be safe to study the impact of NOP14-AS1 on NOP14 expression. NOP14 knockdown using siRNAs had no impact on NOP14-AS1 expression. On the other hand, NOP14 transcriptional repression using CRISPRi resulted in a NOP14-AS1 induction which could not be reversed by an ectopic rescue of NOP14 expression, indicating that this was due to a reduced transcriptional interference. However, the NOP14-AS1 induction observed upon CRISPRi knockdown of NOP14 could not account for the much stronger NOP14-AS1 induction observed upon DNA damage, indicating that an independent mechanism was responsible for the latter. NOP14-AS1 knockdown using antisense oligos / CRISPRi did not have any impact on the NOP14 expression. The DNA damage-induced NOP14 repression could not be reversed upon NOP14-AS1 knockdown. Also, a CRISPRa-mediated NOP14-AS1 induction did not affect NOP14 expression. Together these data indicated that neither the NOP14-AS1 transcript nor its transcription could regulate NOP14 expression. In summary, this study concludes that NOP14-AS1 and NOP14 are independently regulated upon DNA damage. However, the role of NOP14-AS1 in DNA damage remains to be established.

Item Type: Dissertation
Supervisor: Clyaton, Prof. Dr. Christine
Date of thesis defense: 5 May 2017
Date Deposited: 18 May 2017 07:28
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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