CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis.
Nucleic Acids Res
; 44(2): e11, 2016 Jan 29.
Article
em En
| MEDLINE
| ID: mdl-26481349
ABSTRACT
We have developed a new, sequence-specific DNA labeling strategy that will dramatically improve DNA mapping in complex and structurally variant genomic regions, as well as facilitate high-throughput automated whole-genome mapping. The method uses the Cas9 D10A protein, which contains a nuclease disabling mutation in one of the two nuclease domains of Cas9, to create a guide RNA-directed DNA nick in the context of an in vitro-assembled CRISPR-CAS9-DNA complex. Fluorescent nucleotides are then incorporated adjacent to the nicking site with a DNA polymerase to label the guide RNA-determined target sequences. This labeling strategy is very powerful in targeting repetitive sequences as well as in barcoding genomic regions and structural variants not amenable to current labeling methods that rely on uneven distributions of restriction site motifs in the DNA. Importantly, it renders the labeled double-stranded DNA available in long intact stretches for high-throughput analysis in nanochannel arrays as well as for lower throughput targeted analysis of labeled DNA regions using alternative methods for stretching and imaging the labeled long DNA molecules. Thus, this method will dramatically improve both automated high-throughput genome-wide mapping as well as targeted analyses of complex regions containing repetitive and structurally variant DNA.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
DNA
/
Mapeamento Cromossômico
/
Marcação In Situ das Extremidades Cortadas
/
Endonucleases
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
/
Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Estados Unidos