sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing.
Cell Rep
; 26(5): 1098-1103.e3, 2019 01 29.
Article
em En
| MEDLINE
| ID: mdl-30699341
ABSTRACT
Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3' end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA Guia de Cinetoplastídeos
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Motivos de Nucleotídeos
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Sistemas CRISPR-Cas
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Edição de Genes
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Proteína 9 Associada à CRISPR
Limite:
Animals
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article