Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii

摘要: Malaria remains a major global health issue, affecting millions and killing hundreds of thousands people annually. Efforts to break the transmission cycle causal Plasmodium parasite, cure those that are afflicted, rely upon functional characterization genes essential parasite's growth development. These studies often based manipulations parasite genome disrupt or modify gene interest understand its importance function. However, these approaches can be limited by availability selectable markers time required generate transgenic parasites. Moreover, there also is risk disrupting native regulatory elements with introduction exogenous sequences. To address limitations, we have developed CRISPR-RGR, Streptococcus pyogenes (Sp)Cas9-based editing system for utilizes ribozyme-guide-ribozyme (RGR) single guide RNA (sgRNA) expression strategy polymerase II promoters. Using rodent-infectious yoelii, demonstrate both disruptions coding sequence insertions efficiently generated, producing marker-free parasites homology arms as short 80-100 bp. Additionally, find common practice using one sgRNA produce unintended plasmid integration desired locus replacement events, whereas use two sgRNAs results in only editing. Lastly, show CRISPR-RGR used CRISPR interference (CRISPRi) binding catalytically dead SpCas9 (dSpCas9) region upstream interest, resulting position-dependent, but strand-independent reduction expression. This robust flexible facilitates efficient genetic characterizations species.