Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi

Area of Science:

• Entomology
• Genetics
• Molecular Biology

Background:

• Anopheles stephensi, a primary Asian malaria vector, is expanding into Africa, contributing to urban malaria outbreaks.
• Effective control of An. stephensi is crucial for malaria elimination efforts in Africa.
• CRISPR/Cas9 gene drive technology offers a species-specific, environmentally sound, and scalable approach to pest control.

Purpose of the Study:

• To evaluate the efficacy of four RNA Polymerase III promoters in driving gene drive elements in An. stephensi.
• To optimize spatiotemporal control of Cas9 and guide RNA for efficient gene drive implementation.
• To assess homing efficiency and non-homologous end joining (NHEJ) events in somatic tissues.

Main Methods:

• Insertion of a gene drive element into the cd gene of An. stephensi.
• Assessment of four RNA Pol III promoters for their ability to bias inheritance.
• Determination of homing efficiency and analysis of eye phenotype as an indicator of NHEJ.

Main Results:

• All four tested RNA Pol III promoters demonstrated activity in driving the gene drive element.
• Mean inheritance rates reached up to 99.8%, indicating high efficiency.
• A significant influence of the Cas9-bearing grandparent genotype was observed, likely due to maternal Cas9 deposition.

Conclusions:

• The evaluated RNA Pol III promoters are effective in biasing gene drive inheritance in An. stephensi.
• Maternally deposited Cas9 plays a critical role in gene drive efficiency.
• These findings support the potential of CRISPR/Cas9 gene drives for controlling An. stephensi populations and combating malaria.