CRISPR/Cas9-mediated knockout of Tektin 4-like gene (TEKT4L) causes male sterility of Cydia pomonella

Abstract

The sterile insect technique (SIT) is a well-established and environmentally benign method for population control. Identifying genes that regulate insect fertility while preserving growth and development is crucial for implementing a novel SIT-based pest management approach utilizing CRISPR/Cas9 to target these genes for genetic manipulation. Tektin (TEKT), an essential alpha-helical protein pivotal in sperm formation due to its role in cilia and flagella assembly, has garnered attention. In this study, we identified 7 TEKT genes in the testis of Cydia pomonella, a globally invasive fruit pest. Notably, Tektin4-like (TEKT4L) displayed the highest expression level in male adult especially the testes, suggesting its significance in reproductive processes. By utilizing CRISPR/Cas9 technology to knockout TEKT4L, male sterility was induced, showcasing dominant inherited. When wild-type (WT) females mated with TEKT4L^−/− males, eggs laying proceeded normally, but the hatching rate was dramatically reduced, with only 15.49% progressing to the eyespot stage and 68.86% failing to develop normally. The reproductive fitness of TEKT4L^−/− males was robust enough to facilitate the transmission of genetic modifications efficiently within the C.pomonella population, yielding a small number of viable offspring. Subsequent cage trials demonstrated the effectiveness of this population in suppressing laboratory populations of C.pomonella, achieving notable results with a relatively low release ratio (TEKT4L^−/−♂: WT♂: WT♀ = 5:1:5). Consequently, the targeted disruption of the TEKT4L gene holds promise as a fundamental element in a novel pest control strategy against C. pomonella.