Specific Gene Disruption in the Major Livestock Pests<i>Cochliomyia hominivorax</i>and<i>Lucilia cuprina</i>Using CRISPR/Cas9

Paulo, Daniel F.; Williamson, Megan E; Arp, Alex P.; Li, Fang; Sagel, Agustin; Skoda, S.; Sanchez-Gallego, Joel; Vasquez, Mario; Quintero, Gladys; León, Adalberto A Pérez de; Belikoff, Esther J.; Azeredo-Espin, Ana Maria Lima de; McMillan, W. Owen; Concha, Carolina; Scott, Maxwell J.

doi:10.1534/g3.119.400544

Cited by 38 publications

(37 citation statements)

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“…It will be of interest to determine if the latter are important for male reproduction or mating performance, since male mating performance is critical for any genetic control program 5 . Functional analysis of the malespecific genes and other genes of interest such as Chdsx and Chfru will benefit from our recent work developing efficient CRISPR/ Cas9 gene editing technologies in screwworm 54 .…”

Section: Discussionmentioning

confidence: 99%

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs

et al. 2020

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The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

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Section: Discussionmentioning

confidence: 99%

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs

et al. 2020

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“…Further, such a suppression effect can be enhanced if the males carry multiple female-lethal loci on separate chromosomes [33]. For the multiple loci strategy, the All-in-one gene cassette could be inserted into multiple sites using piggy-Bac-mediated transformation, or by precise gene editing mediated by CRISPR/Cas9 [34]. Since the late stage larvae consume most of the larval diet in a mass rearing plant [35], the early female lethal strains could provide significant savings in diet costs by removing the females from the rearing process at the first instar larvae stage.…”

Section: Discussionmentioning

confidence: 99%

An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT

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Background The New World Screwworm fly (NWS), Cochliomyia hominivorax, is an ectoparasite of warm-blooded animals and a major pest of livestock in parts of South America and the Caribbean where it remains endemic. In North and Central America it was eradicated using the Sterile Insect Technique (SIT). A control program is managed cooperatively between the governments of the United States and Panama to prevent the northward spread of NWS from infested countries in South America. This is accomplished by maintaining a permanent barrier through the release of millions of sterile male and female flies in the border between Panama and Colombia. Our research team demonstrated the utility of biotechnology-enhanced approaches for SIT by developing a male-only strain of the NWS. The strain carried a single component tetracycline repressible female lethal system where females died at late larval/pupal stages. The control program can be further improved by removing females during embryonic development as larval diet costs are significant. Results The strains developed carry a two-component system consisting of the Lucilia sericata bottleneck gene promoter driving expression of the tTA gene and a tTA-regulated Lshid proapoptotic effector gene. Insertion of the sex-specifically spliced intron from the C. hominivorax transformer gene within the Lshid gene ensures that only females die when insects are reared in the absence of tetracycline. In several double homozygous two-component strains and in one “All-in-one” strain that had both components in a single construct, female lethality occurred at the embryonic and/or first instar larval stages when raised on diet without tetracycline. Laboratory evaluation for phenotypes that are relevant for mass rearing in a production facility revealed that most strains had fitness characteristics similar to the wild type J06 strain that is currently reared for release in the permanent barrier. Testing of an “All in one” strain under mass rearing conditions showed that the strain maintained the fitness characteristics observed in small-scale rearing. Conclusions The early female lethal strains described here could be selected by the NWS Control Program for testing at large scale in the production facility to enhance the efficiency of the NWS eradication program.

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“…In this context, elucidating the biological function of SCP/TAPS through the use of functional genomics tools such as the CRISPR-cas9 genome-editing system [77] would be a logical next step. Initial success in full knockout of blowfly genes has been achieved [78], Fig. 4 Phylogenetic relationships among curated SCP/TAPS proteins.…”

Section: Discussionmentioning

confidence: 99%

Major SCP/TAPS protein expansion in Lucilia cuprina is associated with novel tandem array organisation and domain architecture

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Background Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites. Methods Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages. Results A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo). Conclusions Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance.

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Specific Gene Disruption in the Major Livestock PestsCochliomyia hominivoraxandLucilia cuprinaUsing CRISPR/Cas9

Cited by 38 publications

References 65 publications

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs

An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT

Major SCP/TAPS protein expansion in Lucilia cuprina is associated with novel tandem array organisation and domain architecture

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