A temperature-sensitive CRISPR-Cas12a system for sterile insect technique.

The sterile insect technique (SIT) reduces population numbers by releasing sterile males that produce non-viable progeny. Specifically, CRISPR/Cas9-based precision-guided SIT (pgSIT) generates sterile males through genetic crosses of two transgenic lines: a Cas9 strain and a guide RNA (gRNA) strain targeting male sterility and female viability or infertility. However, pgSIT requires separate maintenance of the two lines and sorting to obtain sterile males, creating possible challenges for scaling. To overcome this, we propose using Cas12a nuclease, which is inoperative at lower temperatures but active at higher temperatures. Here, we develop a Cas12a-based pgSIT system involving a single strain containing both the Cas12a nuclease and gRNAs to induce male sterility and female lethality. This strain can be maintained as a mixed stock of both sexes and only activated by increasing temperature, producing sterile males after just one generation. By reducing the challenges that arise with maintaining two separate lines, this system could offer a scalable alternative for vector control in combating vector-borne diseases.