CRISPR/Cas technology is increasingly being used as a common methodology in many cancer biology studies due to the ease and convenience of the technique. Precise editing of genomic DNA has been achieved upon repair of CRISPR-induced DNA double-strand breaks (DSBs) by homologous recombination (HR). HR repairs DNA DSBs with high fidelity and therefore, deficiencies in HR result in genome instability. These deficiencies have been
demonstrated in many cancers. RAD51-dependent HR is a very important pathway for repairing DSBs. Previous studies have shown that genome editing using CRISPR technology
relies on the repair of site-specific DNA DSBs induced by the RNA-guided Cas9 endonuclease. Furthermore, previous studies have shown that the efficiency of CRISPR-mediated HR can be improved by the stimulation of HR promoting factors, such as the RAD51 recombinase. Despite the ease and efficient use the CRISPR/Cas technology for genome editing, one limitation is the potential occurrence of associated off-target effects. If CRISPR technology is planned to be used to target cancer cells with defective HR capabilities, will off-target mutations be likely to occur? In order to answer this question, a system was developed in Saccharomyces cerevisiae using green fluorescent protein (GFP) as a reporter to identify off-target CRISPR-induced DSBs. This study set out to test the number of off-target DSBs that could be introduced by CRISPR-induced genome editing in a RAD51-deficient HR model. We were curious whether loss of RAD51-dependent HR would increase the abundance of off-target CRISPR-induced DSBs in mutant yeast strains as compared to those with a functioning
HR-dependent DNA repair pathway. Preliminary findings using this system will be presented.
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Image Processing Pipeline to Compute Homologous Recombination Score
DNA double-strand breaks (DSBs) occur frequently in eukaryotic
cells, and the homologous recombination pathway (HR) is one
of the major pathways required to repair these breaks. However,
tumor cells that are able to repair DSBs are unlikely to die due
to damage incurred by DNA damaging chemotherapies, such as
platinum compounds. While platinum-based therapies have been
effective in treating various cancers, they also carry harsh side
effects, and thus ideally platinum should be used when the probability of treatment resistance is low. HR scores provide a measure
for patients’ tumor’s HR capacity and have been shown to predict
their chemotherapy response and long-term survival. Calculating
this score manually from immunofluorescence microscopy images
for each patient is error-prone and time-consuming. Herein, we
propose an image processing pipeline that takes as input imaging
data from three emission channels (representing nuclei, S-phase
cells, and HR-mediated repair in a tumor slice) from an epifluorescence microscope and computes the HR score. Our open-source
methodology forms a rationale to develop similar approaches in
predicting chemotherapeutic responses and facilitating to make
treatment decisions.
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- NSF-PAR ID:
- 10343950
- Date Published:
- Journal Name:
- 12th International Conference on Biomedical Engineering and Technology (2022), Tokyo, Japan
- Page Range / eLocation ID:
- 51 to 56
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract In germ cells undergoing meiosis, the induction of double strand breaks (DSBs) is required for the generation of haploid gametes. Defects in the formation, detection, or recombinational repair of DSBs often result in defective chromosome segregation and aneuploidies. Central to the ability of meiotic cells to properly respond to DSBs are DNA damage response (DDR) pathways mediated by DNA damage sensor kinases. DDR signaling coordinates an extensive network of DDR effectors to induce cell cycle arrest and DNA repair, or trigger apoptosis if the damage is extensive. Despite their importance, the functions of DDR kinases and effector proteins during meiosis remain poorly understood and can often be distinct from their known mitotic roles. A key DDR kinase during meiosis is ataxia telangiectasia and Rad3‐related (ATR). ATR mediates key signaling events that control DSB repair, cell cycle progression, and meiotic silencing. These meiotic functions of ATR depend on upstream scaffolds and regulators, including the 9‐1‐1 complex and TOPBP1, and converge on many downstream effectors such as the checkpoint kinase CHK1. Here, we review the meiotic functions of the 9‐1‐1/TOPBP1/ATR/CHK1 signaling pathway during mammalian meiosis.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1145/3535694.3535704
