First report on CRISPR/Cas9 mediated editing of the eye colour gene, Tryptophan 2, 3-dioxygenase in egg plant shoot and fruit borer Leucinodes orbonalis Guenée (Lepidoptera: Crambidae)

• Published in: Journal of Asia-Pacific entomology Vol. 26; no. 1; pp. 102031 - 10
• Main Authors: Ashok, Karuppannasamy, Bhargava, Chikmagalur Nagaraja, Prasad Babu, Karakatti, Rohan, Wishard, Manamohan, Malligopal, Rai, Anil, Sanjay, Kumar Pradhan, Parvathy, Madhusoodanan Sujatha, Kennedy, John Samuel, Asokan, Ramasamy
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.03.2023; 한국응용곤충학회
• Subjects: 3-dioxygenase; color; CRISPR-associated endonuclease 9; CRISPR-Cas systems; CRISPR/Cas9; eggplants; entomology; eyes; fruits; genes; Genome editing; Insect eye pigment; Leucinodes orbonalis; mutants; phenotype; ribonucleoproteins; sex determination; spermatogenesis; sterile insect technique; transgenesis; tryptophan; Tryptophan 2; 농학; Genome editing; Leucinodes orbonalis; CRISPR/Cas9; Tryptophan 2; 3-dioxygenase; Insect eye pigment
• ISSN: 1226-8615; 1876-7990
• DOI: 10.1016/j.aspen.2022.102031

[Display omitted] •Developed first-time CRISPR/Cas9 mediated eye colour edit in Leucinodes orbonalis.•Tryptophan 2,3 dioxygenase is the first enzyme of ommochrome pathway in eye colour.•Transcribed gRNA from fifth exon of the above gene, formed RNP complex with Cas9.•RNP was microinjected into G0 stage eggs that resulted reddish brown eyes in adults. Overuse of synthetic chemicals over a long period of time has not only resulted in control failures but also enormous ecological damage. This necessitates developing eco-friendly, effective alternatives for sustainable pest management. In this regard, potential of CRISPR/Cas9 mediated genome editing to introduce site specific mutations that mostly resulting in loss of function has been successfully demonstrated in wide varieties of organisms including insects. This has opened a new avenue to design and implement futuristic pest management strategies like precision guided sterile insect technique (pgSIT) for an area wide suppression. This requires validation of target genes employing Ribonucleoprotein (RNP) complex containing sgRNA and Cas9 protein before undertaking transgenesis for achieving pgSIT. In the present study, we have for the first time, supporting CRISPR/Cas9 mediated editing of the eye colour gene, Tryptophan 2, 3-dioxygenase of the eggplant shoot and fruit borer, Leucinodes orbonalis using RNP complex. The mutant moths of both sexes exhibited reddish brown eyed phenotype. Having established an RNP mediated editing system in L. orbonalis, the key genes involved in sex determination and spermatogenesis will be validated for developing a pgSIT system for L. orbonalis.