CRISPR/CAS9 MEDIATED GENOME EDITING IN MANGO FRUIT FLY, Bactrocera dorsalis WHITE GENE
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Date
2020-12-09
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University of Agricultural Sciences, Bangalore
Abstract
Bactrocera dorsalis is a polyphagous pest capable of making 80-100% yield loss. The dispersive insect needs area wide control measure like precision guided sterile insect technique (PGSIT) comprising CRISPR/Cas9 mediated genome editing. The white gene, contributing brick red eye colour of fruit fly was characterized for optimization of CRISPR/Cas9 mediated target specific editing in the present research. The validation of efficiency and functionality of CRISPR/Cas9 in Bactrocera system was achieved through phenotyping and genotyping the transformed mutant flies. The sgRNA- Cas9 (single
guide- Cas9) complex as ribonucleoprotein (RNP) was delivered through electroporation and microinjection into G0 stage embryos of the flies for integrative transformation. The electroporation with 800 V using sgRNA 443 produced a mutant phenotype fly with blackish green eye colour instead of wild brick red colour. Altered merged and indistinct head spots instead of wild with well distinct and demarcated head spots with a pattern of big spot surrounded by small spots in circle, less melanised body pigmentation and less yellowish thoracic plates were also obtained. The genotype analysis of the white mutant fly has shown a deletion of 3 bp from 449 bp to 452 bp region and variations of two mismatches of nucleotides at 453, 454th position in white gene. Protein analysis revealed differences in the amino acid sequences of the mutant from wild fly, explaining the failure of red eye in the mutant as the protein maybe truncated or non-functional. This optimized protocol can be used for CRISPR/Cas9 mediated knock-down studies in B. dorsalis.