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Site‐specific transgenesis of the Drosophila melanogaster Y‐chromosome using CRISPR/Cas9
Author(s) -
Buchman A.,
Akbari O. S.
Publication year - 2019
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1111/imb.12528
Subject(s) - biology , genetics , drosophila melanogaster , crispr , heterochromatin , chromosome engineering , bacterial artificial chromosome , chromosome , polytene chromosome , cas9 , computational biology , gene , genome
Despite the importance of Y‐chromosomes in evolution and sex determination, their heterochromatic, repeat‐rich nature makes them difficult to sequence (due, in part, to ambiguities in sequence alignment and assembly) and to genetically manipulate. Therefore, they generally remain poorly understood. For example, the Drosophila melanogaster Y‐chromosome, one of the most extensively studied Y‐chromosomes, is widely heterochromatic and composed mainly of highly repetitive sequences, with only a handful of expressed genes scattered throughout its length. Efforts to insert transgenes on this chromosome have thus far relied on either random insertion of transposons (sometimes harbouring ‘landing sites’ for subsequent integrations) with limited success or on chromosomal translocations, thereby limiting the types of Y‐chromosome‐related questions that could be explored. Here, we describe a versatile approach to site‐specifically insert transgenes on the Y‐chromosome in D. melanogaster via CRISPR/Cas9‐mediated homology‐directed repair. We demonstrate the ability to insert, and detect expression from, fluorescently marked transgenes at two specific locations on the Y‐chromosome, and we utilize these marked Y‐chromosomes to detect and quantify rare chromosomal nondisjunction effects. Finally, we discuss how this Y‐docking technique could be adapted to other insects to aid in the development of genetic control technologies for the management of insect disease vectors and pests.

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