In vitro analysis of CRISPR-Cas cleavage dynamics and specificity

Gene therapy is the treatment of inherited disorders, cancer, or infectious diseases, by disrupting the expression of a disease-associated gene or by replacing or correcting a mutated locus in the affected cell type. Besides conventional gene transfer protocols, designer nucleases, such as zinc-fing...

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Autor principal: Gonzalez Perez, Ignacio
Otros Autores: Martí, Marcelo
Formato: Tesis de maestría acceptedVersion
Lenguaje:Inglés
Publicado: Facultad de Farmacia y Bioquímica 2019
Materias:
Análisis in vitro
CRISPR-Cas
Cas9
Cpf1
Gene therapy
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5933
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5933.dir/5933.PDF
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Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_5933
record_format dspace
spelling I28-R145-HWA_59332022-04-19 Gene therapy is the treatment of inherited disorders, cancer, or infectious diseases, by disrupting the expression of a disease-associated gene or by replacing or correcting a mutated locus in the affected cell type. Besides conventional gene transfer protocols, designer nucleases, such as zinc-finger nucleases, TALE nucleases, meganucleases or CRISPR-Cas nucleases, have taken more and more importance in this field. The use of designer endonucleases allows researchers to correct the deleterious mutation in situ by promoting DNA double strand breaks. The CRISPR-Cas technology is being widely used as a powerful genome editing tool because of its simple nature, consisting of a nuclease that is guided to the target site by a single RNA molecule. Nevertheless, one of the major issues is the off-target activity of the CRISPR-Cas system, which is connected to the similarity of other sequences in the genome that are targeted by a particular CRISPR-Cas ribonucleoprotein (RNP) complex. Previous research has demonstrated that besides sequence homology, the off-target activity positively correlates with the RNP concentration in a cell and the time the genome is exposed to these RNPs. Until now, however, not many assays have been performed that characterize in detail the parameters that affect the on and off-target cleavage dynamics. In my Master thesis project, I developed novel in vitro methods to assess the impact of various parameters on both ON and OFF-target cleavage kinetics and cleavage efficiency. I found that ON and OFF target cleavage kinetics and efficiency depend on (i) the target sequence itself, (ii) the ratio between RNPs and target site, (iii) the concentration of RNPs, (iv) the concentration of OFF-target cleavage sites as well as (v) the concentration of OFF-target binding sites. My results imply that CRISPR-Cas DNA cleavage dynamics strongly depend on the concentrations of RNP, DNA target site and RNP OFF-target binding sites. The data suggests that cleavage efficiency in a cell is not only dependent on the target site composition itself but also by the number of OFF-target cleavage sites and ? more importantly ? the number of OFF target binding sites. Fil: Gonzalez Perez, Ignacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Martí, Marcelo Facultad de Farmacia y Bioquímica Cathomen, Toni Gonzalez Perez, Ignacio 2019-03-06 application/pdf Wetzler, Diana Parisi, Gustavo Wäsch, Ralph Análisis in vitro CRISPR-Cas Cas9 Cpf1 Gene therapy eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida In vitro analysis of CRISPR-Cas cleavage dynamics and specificity info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5933 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5933.dir/5933.PDF
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Inglés
orig_language_str_mv eng
topic Análisis in vitro
CRISPR-Cas
Cas9
Cpf1
Gene therapy
Ciencias de la vida
spellingShingle Análisis in vitro
CRISPR-Cas
Cas9
Cpf1
Gene therapy
Ciencias de la vida
Gonzalez Perez, Ignacio
In vitro analysis of CRISPR-Cas cleavage dynamics and specificity
topic_facet Análisis in vitro
CRISPR-Cas
Cas9
Cpf1
Gene therapy
Ciencias de la vida
description Gene therapy is the treatment of inherited disorders, cancer, or infectious diseases, by disrupting the expression of a disease-associated gene or by replacing or correcting a mutated locus in the affected cell type. Besides conventional gene transfer protocols, designer nucleases, such as zinc-finger nucleases, TALE nucleases, meganucleases or CRISPR-Cas nucleases, have taken more and more importance in this field. The use of designer endonucleases allows researchers to correct the deleterious mutation in situ by promoting DNA double strand breaks. The CRISPR-Cas technology is being widely used as a powerful genome editing tool because of its simple nature, consisting of a nuclease that is guided to the target site by a single RNA molecule. Nevertheless, one of the major issues is the off-target activity of the CRISPR-Cas system, which is connected to the similarity of other sequences in the genome that are targeted by a particular CRISPR-Cas ribonucleoprotein (RNP) complex. Previous research has demonstrated that besides sequence homology, the off-target activity positively correlates with the RNP concentration in a cell and the time the genome is exposed to these RNPs. Until now, however, not many assays have been performed that characterize in detail the parameters that affect the on and off-target cleavage dynamics. In my Master thesis project, I developed novel in vitro methods to assess the impact of various parameters on both ON and OFF-target cleavage kinetics and cleavage efficiency. I found that ON and OFF target cleavage kinetics and efficiency depend on (i) the target sequence itself, (ii) the ratio between RNPs and target site, (iii) the concentration of RNPs, (iv) the concentration of OFF-target cleavage sites as well as (v) the concentration of OFF-target binding sites. My results imply that CRISPR-Cas DNA cleavage dynamics strongly depend on the concentrations of RNP, DNA target site and RNP OFF-target binding sites. The data suggests that cleavage efficiency in a cell is not only dependent on the target site composition itself but also by the number of OFF-target cleavage sites and ? more importantly ? the number of OFF target binding sites.
author2 Martí, Marcelo
author_facet Martí, Marcelo
Gonzalez Perez, Ignacio
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Gonzalez Perez, Ignacio
author_sort Gonzalez Perez, Ignacio
title In vitro analysis of CRISPR-Cas cleavage dynamics and specificity
title_short In vitro analysis of CRISPR-Cas cleavage dynamics and specificity
title_full In vitro analysis of CRISPR-Cas cleavage dynamics and specificity
title_fullStr In vitro analysis of CRISPR-Cas cleavage dynamics and specificity
title_full_unstemmed In vitro analysis of CRISPR-Cas cleavage dynamics and specificity
title_sort in vitro analysis of crispr-cas cleavage dynamics and specificity
publisher Facultad de Farmacia y Bioquímica
publishDate 2019
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5933
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5933.dir/5933.PDF
work_keys_str_mv AT gonzalezperezignacio invitroanalysisofcrisprcascleavagedynamicsandspecificity
_version_ 1766017554965331968

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