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

By Andrew R. Bassett, Charlotte Tibbit, Chris P. Ponting, Ji-Long Liu. Cell Reports, 2013

815 citations recorded by the source corpus.

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.

Subfields: Aging, Insect Science, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/261dd296b93905d47c0227e5937c7af9b9dcb14c

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Aging

1. Tzumin Lee

In Molecular Biology

1. Haifeng Wang
2. Marie La Russa
3. Tzumin Lee
4. S. Perry
5. S. Bullock
6. Lei S. Qi
7. Raymond W. M. Kwong
8. A. Zimmer

Citations in the corpus

• Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila (2014, background): “However, experiments in Drosophila so far have failed to detect induced mutations at sites without perfect complementarity (6, 11).”
• The new frontier of genome engineering with CRISPR-Cas9 (2014, neutral): “Efficient genome engineering to allow targeted genome modifications in the germ lines of animal models such as fruit flies (113, 114), zebrafish (94, 115), nematodes (116), salamanders (117), and frogs (118, 119) is now possible with the development of the CRISPR-Cas9 technology.”
• Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila (2014, background): “…HDR with dsDNA donors Most of the initial experiments employing the CRISPR/Cas9 system in Drosophila have depended on easily scored phenotypes for identification of targeted events (Bassett et al. 2013; Gratz et al. 2013a; Kondo and Ueda 2013; Ren et al. 2013; Sebo et al. 2013; Yu et al. 2013).”
• Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing? (2019, methodology): “, 2013), this technique was adopted for studies on fruit flies (Bassett et al., 2013; Gratz et al., 2013), zebrafish (Chang et al.”
• CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “For example, coinjection of Cas9-encoding mRNA and an sgRNA into zygotes or embryos can generate genome-edited animals (36, 128, 248, 249).”