Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei

We developed new P. berghei parasite lines by integrating a dimerisable Cre (DiCre) cassette and a fluorescent marker (GFP or mCherry) in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR-Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked-out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirm the role of CLAMP during invasion of erythrocytes. We used the Oxford Nanopore Technology to sequence the genome of parental (Cas9), Cas-DiCre-GFP, Cas-DiCre-mCherry and CLAMP-cKO parasites.