A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins.
J Vis Exp
; (134)2018 04 30.
Article
em En
| MEDLINE
| ID: mdl-29757293
ABSTRACT
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Ribonucleoproteínas
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Mutação Puntual
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Caenorhabditis elegans
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Genômica
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Sistemas CRISPR-Cas
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article