Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome.
Nat Biotechnol
; 41(4): 488-499, 2023 04.
Article
em En
| MEDLINE
| ID: mdl-36217031
ABSTRACT
Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia Genética
/
Integrases
Limite:
Humans
Idioma:
En
Revista:
Nat Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos