CRISPR/Cas9 BASED EDITING OF SOME IMPORTANT GENES OF MANGO FRUIT FLY, Bactrocera dorsalis (Hendel) (DIPTERA: TEPHRITIDAE)
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Date
2020-11-13
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University of Agricultural Sciences, Bangalore
Abstract
The utility of genome editing in the Bactrocera dorsalis by CRISPR/Cas method was evaluated. We used ribonucleoprotein (RNP) complexes assembled with Cas9 endonuclease as a single guide RNA (sgRNA) engineered with the target pigmentation gene (white) and spermatogenesis (per). The embryos, along with the white gene sgRNA-AS443 (ACCGTTCAGCAAGCGTACGG) (100pmol), Cas9 (400 pmol) and Tris Buffer (10mM) were subjected to electroporation with 800 V@500μs. In contrast to the wild brown reddish eye type,
the developing adult flies displayed a blackish green eye colour with altered head pigmentation.
Compared to the wild style with different spots, all the head spots were indistinct in the edited
type. With lighter pigmentation, the thoracic plate in the edited form was yellowish, while in the
wild type it was more melanized and whitish yellow. In order to obtain more G0 phased embryo
numbers, optimization of egg laying was carried out, which showed that the banana pulp was B.
dorsalis most favoured oviposition substrate. There was no significant difference between water
media and 0.5% solidified agar media for egg processing, while after incubation, survivability in
water media was found to be higher. The peak time of egg laying was found in the 21-43-day
(23.93±10.10 eggs) age range. Five flies per box with an average number of eggs of 162.83±75.80 in the 1st hour, 107±92.19 in the 2nd hour and 142.66±29.27 in the 3rd hour is deemed better handling. The research reveals that synthetic RNP complexes constructed in vitro can be used in studies of genetic manipulation.