Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

• Published in: Scientific reports Vol. 8; no. 1; pp. 13125 - 14
• Main Authors: Waters, Andrew J., Capriotti, Paolo, Gaboriau, David C. A., Papathanos, Philippos Aris, Windbichler, Nikolai
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03-09-2018; Nature Publishing Group
• Subjects: 38/39; 38/77; 42/41; 42/44; 631/208/199; 631/553/552; 631/61; 64/24; Animal species; Animals; Base Sequence; Biotechnology; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; Drosophila melanogaster; Drosophila melanogaster - genetics; Drosophila Proteins - genetics; Economic importance; Evolutionary conservation; Female; Gene Editing - methods; Genes, Insect; Genes, Lethal; Genetic control; Genetic engineering; Genetic Fitness; Genetic isolation; Genetic Loci; Genetically engineered organisms; Genome editing; Genome, Insect; Genomes; Homeodomain Proteins - genetics; Humanities and Social Sciences; INDEL Mutation; Insects; Lethality; Male; multidisciplinary; Plant species; Population Control - methods; Promoter Regions, Genetic; Reproductive fitness; Reproductive Isolation; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Science; Science (multidisciplinary); Sequence Alignment; Transcription; Transcription Factors - genetics; Transcriptional Activation
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-31433-2

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model
Drosophila melanogaster
by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the
eve
locus and find that they disrupt development by strongly activating
eve
outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.