Raw images of agarose gel electrophoresis.

<div><p>Malaria caused by <i>Plasmodium</i> parasites remains a large health burden. One approach to combat this disease involves vaccinating individuals with whole sporozoites that have been genetically modified to arrest their development at a specific stage in the liver by targeted gene deletion, resulting in a genetically attenuated parasite (GAP). Through a comprehensive phenotyping screen, we identified the <i>hscb</i> gene, encoding a putative iron-sulfur protein assembly chaperone, as crucial for liver stage development, making it a suitable candidate gene for GAP generation. Parasites lacking <i>Plasmodium berghei</i> HscB (<i>Pb</i>HscB) exhibited normal sporozoite production in mosquitoes, but their liver stage development was severely impaired, characterized by slow growth and delayed expression of merozoite surface protein 1 (MSP1). <i>In vivo</i> experiments demonstrated that <i>Pb</i>HscB-deficient parasites exhibited a delay in prepatency of 2–4 days, emphasizing the significance of <i>Pb</i>HscB for exo-erythrocytic development. Although knockout of <i>Pb</i>HscB alone allowed breakthrough infections, it is a potent candidate for a dual gene deletion strategy. PlasMei2, an RNA-binding protein, was previously found to be crucial for the completion of liver stage development. We generated a <i>Pb</i>HscB-<i>Pb</i>Mei2-double attenuated parasite line, serving as a late liver stage-arresting replication-competent (LARC) GAP, providing a solid block of liver-to-blood stage transition.</p></div>