Ribozyme-Mediated, Multiplex CRISPR Gene Editing and CRISPRi in Plasmodium yoelii

摘要: Functional characterization of genes in Plasmodium parasites often relies on genetic manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by the time required generate transgenic for P. falciparum and availability single drug selectable marker yoelii. In both cases, there remains risk disrupting native gene regulatory elements with introduction exogenous sequences. To address limitations, we have developed CRISPR-RGR, SpCas9-based editing system that utilizes Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. Using this yoelii, demonstrate disruptions coding sequence insertions efficiently generated, producing marker-free scar-free homology arms as short 80-100bp. Moreover, find common practice using one can produce unintended plasmid integration desired locus replacement events, while use two sgRNAs results only editing. Lastly, show CRISPR-RGR be used CRISPR interference (CRISPRi) binding dCas9 targets control region gene-of-interest, resulting modest reduction expression. This robust flexible should open door in-depth efficient characterizations rodent- human-infectious species.