Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.
Cell Rep
; 9(3): 1151-62, 2014 Nov 06.
Article
em En
| MEDLINE
| ID: mdl-25437567
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Guia de Cinetoplastídeos
/
Drosophila melanogaster
/
Proteínas Associadas a CRISPR
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Limite:
Animals
Idioma:
En
Revista:
Cell Rep
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
China