eprintid: 27932
rev_number: 8
eprint_status: archive
userid: 4997
dir: disk0/00/02/79/32
datestamp: 2020-03-04 13:32:33
lastmod: 2020-03-13 10:31:47
status_changed: 2020-03-04 13:32:33
type: doctoralThesis
succeeds: 27931
metadata_visibility: show
creators_name: Roidos, Paris
title: Development of a high-throughput CRISPR/Cas9 based fluorescent reporter to study DNA double-strand break repair choices.
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
keywords: CRISPR/Cas9 based assessment of DNA DSB repair choices.
cterms_swd: DNA repair
cterms_swd: CRISPR/Cas9
cterms_swd: CAT-R fluorescent reporter
abstract: DNA double-stranded breaks (DSBs) are the most toxic types of DNA lesions. Cells have several strategies to repair such lesions that can be grouped into end-protection and end-resection coupled mechanisms. To study the DNA DSB choices in-vitro, we developed and employed Color-Assay-Tracing-Repair (CAT-R) as a dual-fluorescence reporter taking advantage of the highly efficient CRISPR/Cas9 system to induce DSBs. We can distinguish point-mutations (InDels) from large-deletions/insertions in-vitro and quantify simultaneously the rates of NHEJ vs end-resection based DNA repair. We combined this system with high-throughput flow cytometry and studied the DNA DSB repair choices. On the one hand, we evaluated the efficiency and potency of 26 different pharmacological compounds, that are currently in clinical/preclinical studies targeting ATM, DNA-PK, ATR, and PARP, and we identify key differences in the way these compounds engage to DNA DSB repair choice. On the other hand, in order to find new players that can regulate the choice between end-protection and end-resection we performed a custom designed CRISPR/Cas9 arrayed genetic screen with CAT-R and evaluated the functions of 420 individual DNA repair components. For this, we developed a highly efficient transfection platform for arrayed CRISPR/Cas9 screens based on solid phase transfection with synthetic gRNA:tracrRNA complexes. In addition to known components of DNA DSBs we uncovered novel molecules that can tip the balance of the DNA DSB repair choice either towards end-protection or end-resection. In summary, CAT-R can be used to assess the functions of DNA DSB repair components in genetic/chemical screens in a variety of mammalian cells.
date: 2020
id_scheme: DOI
id_number: 10.11588/heidok.00027932
ppn_swb: 1692441493
own_urn: urn:nbn:de:bsz:16-heidok-279321
date_accepted: 2020-02-10
advisor: HASH(0x55e83b577478)
language: eng
bibsort: ROIDOSPARIDEVELOPMEN2020
full_text_status: public
place_of_pub: Heidelberg
citation:   Roidos, Paris  (2020) Development of a high-throughput CRISPR/Cas9 based fluorescent reporter to study DNA double-strand break repair choices.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/27932/1/PhD.Thesis-P.Roidos.pdf