Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters.
Nat Commun
; 6: 8101, 2015 Sep 01.
Article
em En
| MEDLINE
| ID: mdl-26323354
ABSTRACT
The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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DNA Bacteriano
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Família Multigênica
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Cromossomos Bacterianos
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Clonagem Molecular
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Endonucleases
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article