Publicação

Development of TAL effector: recombinase fusion proteins for genome engineering

Detalhes bibliográficos
Resumo:	Genome engineering is currently entering the phase of genetic manipulation in vivo. Several important discoveries have made this progress possible, such as the development of site-specific recombinases (SSRs) and the discovery of TAL effector (TALE) proteins a novel type of DNA-binding proteins. SSRs have proved to be reliable and widely used engineering tools to manipulate DNA in vitro and in vivo. They posses unique ability to mediate efficient and precise integration, deletion or inversion of defined DNA segments. Hyperactivated variants of the resolvase/invertase family of serine recombinases do not required accessory factors for recombination. Thereby they can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered TALE proteins, generating a chimeric TALE recombinase (TALER) with programmable sequence specificity. Here we described a chimeric TALE recombinase assembled in order to mediate site-specific recombination on novel sequences. We engineered a fusion between a hyperactivated catalytic domain from the Tn3 resolvase and a functional DBD assembled utilizing the customized TALE design. We use three different truncated TALE variants to generate diverse TALER constructs and six different target sites. TALE domain was assembled and tested beforehand through its fusion with a nuclease protein. We have shown that TALE DNA-binding domain works efficiently and when fused to a nonspecific cleavage domain is able to introduce DNA strand breaks at specific genomic sequence in human cells. We also show that current designs of TALERs did not mediate site-specific recombination on the predicted target sites when tested in bacterial cells. We discuss some reasons that might be relevant for the obtain results. Although TALER fusions described herein did not produce a functional variant. This work demonstrates that further optimization of several technical details can be made. The creation of novel recombinases domains promises significantly expanding the targeting capacity of engineered recombinases in genome engineering and even gene therapy treatments.
Autores principais:	Pina, Lígia Marisa Sampaio, 1990-
Assunto:	Engenharia genética Recombinação genética Transcrição genética Teses de mestrado - 2013
Ano:	2013
País:	Portugal
Tipo de documento:	dissertação de mestrado
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade de Lisboa
Idioma:	inglês
Origem:	Repositório da Universidade de Lisboa

Descrição
Resumo:	Genome engineering is currently entering the phase of genetic manipulation in vivo. Several important discoveries have made this progress possible, such as the development of site-specific recombinases (SSRs) and the discovery of TAL effector (TALE) proteins a novel type of DNA-binding proteins. SSRs have proved to be reliable and widely used engineering tools to manipulate DNA in vitro and in vivo. They posses unique ability to mediate efficient and precise integration, deletion or inversion of defined DNA segments. Hyperactivated variants of the resolvase/invertase family of serine recombinases do not required accessory factors for recombination. Thereby they can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered TALE proteins, generating a chimeric TALE recombinase (TALER) with programmable sequence specificity. Here we described a chimeric TALE recombinase assembled in order to mediate site-specific recombination on novel sequences. We engineered a fusion between a hyperactivated catalytic domain from the Tn3 resolvase and a functional DBD assembled utilizing the customized TALE design. We use three different truncated TALE variants to generate diverse TALER constructs and six different target sites. TALE domain was assembled and tested beforehand through its fusion with a nuclease protein. We have shown that TALE DNA-binding domain works efficiently and when fused to a nonspecific cleavage domain is able to introduce DNA strand breaks at specific genomic sequence in human cells. We also show that current designs of TALERs did not mediate site-specific recombination on the predicted target sites when tested in bacterial cells. We discuss some reasons that might be relevant for the obtain results. Although TALER fusions described herein did not produce a functional variant. This work demonstrates that further optimization of several technical details can be made. The creation of novel recombinases domains promises significantly expanding the targeting capacity of engineered recombinases in genome engineering and even gene therapy treatments.