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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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Inglés |
| Publicado: |
Facultad de Farmacia y Bioquímica
2019
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| 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 |
| Aporte de: |
| Sumario: | 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. |
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