Genome editing tool for functional characterization of variants of unknown significance (VUS)
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/90339 |
Resumo: | Familial breast cancer accounts for almost 15% of all breast cancer cases, and it is predominantly due to inherited mutations on BRCA1 and BRCA2 genes. Some other genes have also been described to be linked to breast cancer development, as well as, with Homologous Recombination (HR), which is one of the main pathways to repair DNA double-strand breaks (DSBs). In recent years, many advances have been made in sequencing, through the widespread of next-generation sequencing (NGS), each clinical panel has been extended to numerous genes, and since genetic testing has become standard, there has been an increased detection of variants of unknown significance (VUS) without established clinical guidelines. These VUS can either be pathogenic or benign, but since their effect is unclear, functional assays need to be performed to categorise their mutational status. Nevertheless, there is still a need to take the patient’s blood in order to perform such assays. Hence, by using a genome-editing tool, such as CRISPR-Cas9, we would be able to introduce the VUS found in the patients into a non-tumorigenic breast cell line, MCF10-A. CRISPR-Cas9 methodology is based on the sgRNA recruitment of the Cas9 nuclease to the specific target locus and, by complementarity, the induction of a DSB. These can be repaired either by HR, which is dependent on a repair template (single-stranded DNA oligonucleotides – ssODN) or NHEJ, which is error-prone. So, the first step was to establish our cellular model by introducing the sgRNA and ssODN, previously created through a CRISPR design tool and place them into a pSpCas9(BB) plasmid, pX458. After constructing the pX458 sgRNA ssODN, it was transfected into the MCF10-A cells, where single-cell clone isolation and selection was performed by flow cytometry. Afterwards, we verified if the desired point mutation was indeed inserted through PCR and sequencing. The intent was to create three cell lines: the non-tumorigenic MCF10-A, negative control; MCF10-A homozygous for the VUS, positive control and the one which would mimic the patients the best, MCF10-A heterozygous for the VUS. These cells would undergo a genotoxic challenge using three different drugs, doxorubicin (DOX), hydrogen peroxide (H2O2) and PARP inhibitors (PARPi) to assess and compare the DNA damage induced in the three cell lines through functional assays, such as comet assay, which measures low levels of DNA damage, and ɣ-H2AX flow cytometry, which measures DNA DSBs. The cell lines with VUS, homozygous and heterozygous, have not been established yet, so we have only evaluated the DNA damage in the non-tumorigenic MCF10-A cell line. The results are as expected, in the comet assay H2O2 50μM, the positive control, has the highest %DNA in tail, followed by DOX 5μM. Both DOX 0.1μM and DOX 1μM are not statistically different from the negative control (no drug added). On the other hand, MCF10-A cells have increased sensitivity to DOX 1μM and DOX5μM (positive control), since they have higher %ɣ-H2AX, which means they originate more DNA DSBs, whereas none of H2O2 nor PARPi concentrations present significant differences from the negative control. Nevertheless, we still need to establish the MF10-A cells with the VUS in order to understand their function, as well as their role on cancer risk. |
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Genome editing tool for functional characterization of variants of unknown significance (VUS)Familial Breast CancerVariants of Unknown SignificanceBRCA1 and BRAC2CRISPR-Cas9MCF10-A cellsChemotherapeutic agentsDomínio/Área Científica::Ciências MédicasFamilial breast cancer accounts for almost 15% of all breast cancer cases, and it is predominantly due to inherited mutations on BRCA1 and BRCA2 genes. Some other genes have also been described to be linked to breast cancer development, as well as, with Homologous Recombination (HR), which is one of the main pathways to repair DNA double-strand breaks (DSBs). In recent years, many advances have been made in sequencing, through the widespread of next-generation sequencing (NGS), each clinical panel has been extended to numerous genes, and since genetic testing has become standard, there has been an increased detection of variants of unknown significance (VUS) without established clinical guidelines. These VUS can either be pathogenic or benign, but since their effect is unclear, functional assays need to be performed to categorise their mutational status. Nevertheless, there is still a need to take the patient’s blood in order to perform such assays. Hence, by using a genome-editing tool, such as CRISPR-Cas9, we would be able to introduce the VUS found in the patients into a non-tumorigenic breast cell line, MCF10-A. CRISPR-Cas9 methodology is based on the sgRNA recruitment of the Cas9 nuclease to the specific target locus and, by complementarity, the induction of a DSB. These can be repaired either by HR, which is dependent on a repair template (single-stranded DNA oligonucleotides – ssODN) or NHEJ, which is error-prone. So, the first step was to establish our cellular model by introducing the sgRNA and ssODN, previously created through a CRISPR design tool and place them into a pSpCas9(BB) plasmid, pX458. After constructing the pX458 sgRNA ssODN, it was transfected into the MCF10-A cells, where single-cell clone isolation and selection was performed by flow cytometry. Afterwards, we verified if the desired point mutation was indeed inserted through PCR and sequencing. The intent was to create three cell lines: the non-tumorigenic MCF10-A, negative control; MCF10-A homozygous for the VUS, positive control and the one which would mimic the patients the best, MCF10-A heterozygous for the VUS. These cells would undergo a genotoxic challenge using three different drugs, doxorubicin (DOX), hydrogen peroxide (H2O2) and PARP inhibitors (PARPi) to assess and compare the DNA damage induced in the three cell lines through functional assays, such as comet assay, which measures low levels of DNA damage, and ɣ-H2AX flow cytometry, which measures DNA DSBs. The cell lines with VUS, homozygous and heterozygous, have not been established yet, so we have only evaluated the DNA damage in the non-tumorigenic MCF10-A cell line. The results are as expected, in the comet assay H2O2 50μM, the positive control, has the highest %DNA in tail, followed by DOX 5μM. Both DOX 0.1μM and DOX 1μM are not statistically different from the negative control (no drug added). On the other hand, MCF10-A cells have increased sensitivity to DOX 1μM and DOX5μM (positive control), since they have higher %ɣ-H2AX, which means they originate more DNA DSBs, whereas none of H2O2 nor PARPi concentrations present significant differences from the negative control. Nevertheless, we still need to establish the MF10-A cells with the VUS in order to understand their function, as well as their role on cancer risk.Silva, Susana Nunes daRUNPires, Maria João Almeida e Silva2022-12-06T01:30:52Z2019-12-042019-09-262019-12-04T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/90339TID:202350924enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T04:40:12Zoai:run.unl.pt:10362/90339Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:37:08.592816Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
title |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
spellingShingle |
Genome editing tool for functional characterization of variants of unknown significance (VUS) Pires, Maria João Almeida e Silva Familial Breast Cancer Variants of Unknown Significance BRCA1 and BRAC2 CRISPR-Cas9 MCF10-A cells Chemotherapeutic agents Domínio/Área Científica::Ciências Médicas |
title_short |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
title_full |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
title_fullStr |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
title_full_unstemmed |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
title_sort |
Genome editing tool for functional characterization of variants of unknown significance (VUS) |
author |
Pires, Maria João Almeida e Silva |
author_facet |
Pires, Maria João Almeida e Silva |
author_role |
author |
dc.contributor.none.fl_str_mv |
Silva, Susana Nunes da RUN |
dc.contributor.author.fl_str_mv |
Pires, Maria João Almeida e Silva |
dc.subject.por.fl_str_mv |
Familial Breast Cancer Variants of Unknown Significance BRCA1 and BRAC2 CRISPR-Cas9 MCF10-A cells Chemotherapeutic agents Domínio/Área Científica::Ciências Médicas |
topic |
Familial Breast Cancer Variants of Unknown Significance BRCA1 and BRAC2 CRISPR-Cas9 MCF10-A cells Chemotherapeutic agents Domínio/Área Científica::Ciências Médicas |
description |
Familial breast cancer accounts for almost 15% of all breast cancer cases, and it is predominantly due to inherited mutations on BRCA1 and BRCA2 genes. Some other genes have also been described to be linked to breast cancer development, as well as, with Homologous Recombination (HR), which is one of the main pathways to repair DNA double-strand breaks (DSBs). In recent years, many advances have been made in sequencing, through the widespread of next-generation sequencing (NGS), each clinical panel has been extended to numerous genes, and since genetic testing has become standard, there has been an increased detection of variants of unknown significance (VUS) without established clinical guidelines. These VUS can either be pathogenic or benign, but since their effect is unclear, functional assays need to be performed to categorise their mutational status. Nevertheless, there is still a need to take the patient’s blood in order to perform such assays. Hence, by using a genome-editing tool, such as CRISPR-Cas9, we would be able to introduce the VUS found in the patients into a non-tumorigenic breast cell line, MCF10-A. CRISPR-Cas9 methodology is based on the sgRNA recruitment of the Cas9 nuclease to the specific target locus and, by complementarity, the induction of a DSB. These can be repaired either by HR, which is dependent on a repair template (single-stranded DNA oligonucleotides – ssODN) or NHEJ, which is error-prone. So, the first step was to establish our cellular model by introducing the sgRNA and ssODN, previously created through a CRISPR design tool and place them into a pSpCas9(BB) plasmid, pX458. After constructing the pX458 sgRNA ssODN, it was transfected into the MCF10-A cells, where single-cell clone isolation and selection was performed by flow cytometry. Afterwards, we verified if the desired point mutation was indeed inserted through PCR and sequencing. The intent was to create three cell lines: the non-tumorigenic MCF10-A, negative control; MCF10-A homozygous for the VUS, positive control and the one which would mimic the patients the best, MCF10-A heterozygous for the VUS. These cells would undergo a genotoxic challenge using three different drugs, doxorubicin (DOX), hydrogen peroxide (H2O2) and PARP inhibitors (PARPi) to assess and compare the DNA damage induced in the three cell lines through functional assays, such as comet assay, which measures low levels of DNA damage, and ɣ-H2AX flow cytometry, which measures DNA DSBs. The cell lines with VUS, homozygous and heterozygous, have not been established yet, so we have only evaluated the DNA damage in the non-tumorigenic MCF10-A cell line. The results are as expected, in the comet assay H2O2 50μM, the positive control, has the highest %DNA in tail, followed by DOX 5μM. Both DOX 0.1μM and DOX 1μM are not statistically different from the negative control (no drug added). On the other hand, MCF10-A cells have increased sensitivity to DOX 1μM and DOX5μM (positive control), since they have higher %ɣ-H2AX, which means they originate more DNA DSBs, whereas none of H2O2 nor PARPi concentrations present significant differences from the negative control. Nevertheless, we still need to establish the MF10-A cells with the VUS in order to understand their function, as well as their role on cancer risk. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-12-04 2019-09-26 2019-12-04T00:00:00Z 2022-12-06T01:30:52Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/90339 TID:202350924 |
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http://hdl.handle.net/10362/90339 |
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TID:202350924 |
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eng |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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