Logo: to the web site of Uppsala University

uu.sePublications from Uppsala University
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Compounds screening for the identification of novel drug to improve the Knock in efficiency mediated by CRISPR-Cas9
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. (AstraZeneca AB R&D, Discovery Biology)
2023 (English)Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
Abstract [en]

Genome editing is an exciting field that allows for the precise modification of an organism's DNA. One of the most advanced tools in this area is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which creates a DSB (Double-strand break) at a specific location in the genome. This break can then be repaired by the cell using one of two pathways – NHEJ (nonhomologous end joining) or HDR (homology-directed repair) HDR leads to more precise repair and is used to create KI (Knock-In) modifications by introducing a homologous piece of DNA with the desired changes. However, HDR is a rare event that competes with the error prone NHEJ pathway, limiting its efficiency. HDR mainly occurs in the G2 and S phases of the cell cycle, making it a challenge to control and target. To improve KI efficiency, researchers have used strategies such as inhibiting NHEJ or activating HDR. This study focuses on identifying direct and indirect activators of HDR through a library assay screening. We established a robust method for screening compounds in HEK293 cells that relies on a plasmid-based delivery Cas9, gRNA (guide RNA), and synthetic ssDNA (single strand DNA). Out of 3,000 compounds screened, 1% showed a higher signal than the positive control, and approximately 10% presented a higher signal than untreated cells. The top 5 compounds were further validated in dose response. Our system opens new avenues for improving the efficiency of KI modifications.

Place, publisher, year, edition, pages
2023.
Keywords [en]
Genome editing, Knock in, HDR, CRISPR-Cas9, screening, NHEJ
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-507498OAI: oai:DiVA.org:uu-507498DiVA, id: diva2:1780835
Educational program
Master Programme in Biology
Presentation
2023-06-07, 13:40 (English)
Supervisors
Examiners
Available from: 2023-08-07 Created: 2023-07-06 Last updated: 2023-08-07Bibliographically approved

Open Access in DiVA

No full text in DiVA

By organisation
Biology Education Centre
Cell and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 300 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf