CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus

Abstract

The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model.

Allele-specific genome editing using CRISPR–Cas9 is associated with loss of heterozygosity in diploid yeast

“…Chromosome-arm LOH has previously been reported upon introduction of a DSB in one of two homologous chromosomes, but was considered rare and has not been described as disruptive to gene-editing approaches (9,13,32). To investigate the extent and nature of the LOH caused by Cas9-editing of heterozygous loci, a strain with an average of four heterozygous SNPs or INDELs per kbp was generated by mating IMX1555 (CEN.PK genetic background, expressing Cas9, mRuby2 and mTurquoise2 from chromosome V) with S288C (Supplementary Table S6).…”

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

“…Finally, we would like to make a point that the gene targeting protocol presented in this study is simpler and more feasible than those published in other studies for the following reasons: (1) lab-produced Cas9 protein is significantly cheaper than commercially available products; (2) pre-assembled Cas9 and gRNAs complexes are likely to be more stable in embryos than, for instance, Cas9 mRNA; (3) Cas9 mRNA production is time consuming and expensive, and can be stored only for few weeks, while purified Cas9 protein can be stored at −80 °C for years, and pre-assembled RNPs can be stored at -80 °C for at least 10 weeks without losing their activity; (4) finally, pre-assembled RNP complexes may have less off target effects 39 , 50 . Our protocol and the purified Cas9 protein have been already successfully applied in the housefly, Musca domestica , to target the Drosophila yellow ortholog brown body 58 and the male determining gene Mdmd 59 .…”

Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing

“…Since the first application of the Clustered Regularly Interspaced Short Palindromic Repeats and its associated endonuclease 9 (CRISPR-Cas9 system) for site-specific genome editing, the technology has been used in a variety of arthropod species 1 , 2 . Successful application of CRISPR-Cas9 to edit the germline of arthropods relies on injection of gene-editing materials into pre-blastoderm embryos (embryonic microinjection) 3 – 6 . This dependence is a significant barrier to the successful general application of transformation technologies, particularly for non-specialist laboratories, as it requires expensive equipment and training to implement 4 .…”