Although genome-editing-based breeding has advanced rapidly in aquaculture, progress in marine mollusks has lagged behind. The main challenge is not genome editing itself, but rather rearing the few edited larvae through metamorphosis to establish a viable G0 founder population.

Researchers from the Institute of Oceanology of the Chinese Academy of Sciences (IOCAS) overcame several technical hurdles, including the difficulty of culturing genome-edited larvae and low metamorphosis rates. They produced a G0 genome-edited population of approximately 100 juvenile Pacific abalones (Haliotis discus hannai). The largest individuals reached approximately three centimeters in shell length. 

Their findings were published in Aquaculture on October 20.

The researchers targeted Hdh-tyr1, a tyrosinase gene associated with shell formation and obtained genome-edited larvae that carried mutations. Rearing these larvae, however, was proved to be the most difficult part of the process. In the early stages of the study, all batches of genome-edited larvae died, largely due to limitations in existing culture techniques.

In conventional small-volume culture systems, protists always outbreak and wipe out entire larval batches within days. After repeated optimization, the researchers reared each larva separately in a single well, which they described as "single apartments." They combined this method with an optimized metamorphosis induction protocol, ensuring larval survival rates higher than 90%.

At the juvenile stage, the researchers identified three benthic diatom species that served as high-quality feed for newly metamorphosed abalones and supported rapid growth. The researchers also developed a simplified DNA extraction method that allowed for the efficient genotyping of very small juveniles.

"The individual-level verification provided an early 'identity verification' of the edited animals," said YU Yanan, the first author of the study. "It also helped confirm that mutated larvae had developed into mutated juveniles."

The resulting workflow integrates the construction of the editing system, the rearing of larvae and juveniles, the induction of metamorphosis, and the genotyping of individual organisms. "Using this workflow, we generated the G0 genome-edited population," said Prof. HUAN Pin, one of the corresponding authors of the study. 

This study represents a significant step toward completing the technical process for genome editing-based breeding in mollusks.