A sensitive and rapid visual method of chicken sexing based on LAMP-CRISPR/Cas12a system.

1. Accurate sex identification of one-day-old chicks is crucial in layer poultry production. Establishing an early sexing method during the chicken embryonic period is essential for animal welfare. However, PCR-based sexing has limitations in terms of specialised equipment and is time-consuming.2. This study presents a rapid, simple and fluorescent visual technique for chicken sex identification based on Loop-mediated isothermal amplification (LAMP)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). It targets the chicken Z chromosome gene DMRT1 and W chromosome-specific fragment EE0.6 using designed primers and sgRNA. The LAMP amplicon is cleaved by Cas12a, producing a fluorescent product detectable by a portable light apparatus.3. The method has high sensitivity, capable of detecting as few as two copies per microlitre of the EE0.6 template and 20 copies per microlitre of the DMRT1 template. This has significant potential for distinguishing chicken embryo gender very early in embryonic development.