Y-chromosome transgenics using CRISPR/Cas9

Lefthiris Zorbas is PhD candidate at the University of Thessaly, Larissa where he is studying the Y chromosome of Bactrocera oleae and its gene content (isolation and characterization of genes and especialy of the maleness factor). Creation of genetic sexing strains of B. oleae for use at the SIT. MORE ABOUT THE AUTHOR

Buchman et al (2018) from Tata Institute of California University reported in Insect Molecular Biology the use of CRISPR/Cas9-HDR system in order to insert transgenes to specific sites of the Drosophila melanogaster Y chromosome. In addition they investigated its usage in identifying rare progeny resulting from meiotic sex chromosome nondisjunction events in females.

Due to its heterochromatic, repetitive and degenerate nature, the Y chromosome is a difficult target to sequence and analyze genomicaly. Additionally, its nature renders it refractory for inserting and/or expressing transgenes. All these factors make Y chromosome manipulation difficult and reliant on random and rare events such as transposone-mediated integration.

Drosophila melanogaster chromosomes. Image Credit:A Short History and Description of Drosophila melanogaster Classical Genetics: Chromosome Aberrations, Forward Genetic Screens, and the Nature of Mutations View ORCID ProfileThomas C. Kaufman Genetics June 1, 2017 vol. 206 no. 2 665-689

The first part of the project was to create a line that would have a site-specific Y chromosome transgene construct insertion. The engineering logic behind this construct is very simple; a td-Tomato fluorescent protein gene under the regulatory control of the 3xP3 eye-specific promoter is flanked by the gypsy and CTCF insulators and serves as a genetic marker. Cloning sites upstream and downstream of the marker gene are sites for inserting homology arm sequences used in subsequent knock-in efforts by CRISPR/Cas9 mediated homology dependent repair. The homology arms were designed to be homologous to specific sites on the Y chromosome where they wanted to perform the insertion.

Insulators can protect transgenes from heterochromatin-based gene silencing. Image Credit: Singer, S.D., Liu, Z., & Cox, K.D. (2011). Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators. Plant Cell Reports, 31, 13-25.

Buchman et al. selected ten different target sites spanning throughout the Y chromosome and they designed sgRNAs for each of them. After site selection and vectors preparation, each transgene was injected along with sgRNA and Cas9 to embryos also expressing Cas9 in the germ line from an integrated vasa-Cas9 transgene for higher efficiency. Only two of the ten constructs injected yielded successful results. Transgenic G1 male progeny was outcrossed with w-virgin females in order to establish the new line carrying the transgene.

Drosophila melanogaster

In the second part of the project Buchman et al performed crosses of the newly created Y-transgenic line with X-balancer line. The X-balancer line contains X chromosomes with abnormalities that lead to nondisjunction events which result in progenies with XXY and XO genotypes. Following the crossings, successful identification of XXY females and XO males was conducted through the presence or absence of red fluorescence respectively.

This study provided a proof of principle example for the successful use of CRISPR/Cas9 on Y chromosomes that, in principle, could be applicable and to other insects, and herald a new era on Y chromosome manipulation and research.

Buchman, A. and Akbari, O. S. (2018), Site‐specific transgenesis of the D. melanogaster Y‐chromosome using CRISPR/Cas9. Insect Mol Biol. Accepted Author Manuscript. . doi:10.1111/imb.12528

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