Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

Summary

Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

Graphical Abstract

Highlights

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  Targeted mutagenesis with CRISPR/Cas9 nucleases of Drosophila genes

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  Highly efficient mutant generation in up to 88% of injected flies

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  Transmission of mutants through the germline, in up to 34.5% of offspring

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  Highly sensitive detection of mutations by high-resolution melt analysis

Abstract

Bassett, Liu, and colleagues present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). They show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline, and this method offers the opportunity to generate a desired mutant fly line within one month.