Genetically attenuated parasites (GAP’s) are effective malaria vaccine candidates in preclinical testing, but have relied upon disrupting essential genes to prevent growth of the parasite in the liver. If these genes are also essential to earlier points in the life cycle, it is not feasible to use them as vaccine candidates, because the parasite would die before forming the necessary parasite stage to infect the host and act as a vaccine. Creating GAP’s by using the CRISPR/Cas9 system to target multiple essential genes-of-interest simultaneously has a promising future. This system has adaptable specificity provided by single guide RNAs (sgRNAs) and the sequence of the sgRNAs is easily manipulated. These CRISPR-GAP’s would be able to grow and successfully infect the host, but the development of the parasite would be hindered without the targeted essential genes, enabling the host to develop immunity toward the entire parasite. CRISPR/Cas9 is a system used by bacteria to recognize and destroy invading phage DNA, which are recognized and cut by a guide RNA bound to the Cas9 nuclease. This system can be manipulated to target essential genes in a parasite by creating single guide RNAs (sgRNA) that target a desired sequence. Constitutive RNA Polymerase III (RNAP III) promoters are typically used to produce sgRNAs, but are not regulatable. Here, ribozymes will be used to cut sgRNAs from RNAP II transcripts in order to target multiple genomic loci, thus allowing for regulatable expression. This strategy of sgRNA expression by a stage-specific promoter enables stage-specific gene editing of Plasmodium parasites.
