falciparum uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves 17 this system was first established in Apicomplexan parasites in Toxoplasma gondii 18 and then adapted successfully to P. The inducible Cre recombinase (DiCre) established for use in P. The use of Cre- and FLP-recombinase has been described in Plasmodium spp. In other organisms site-specific recombinases are widely used to modify genomes, for example the total removal of a target gene or an important part thereof. falciparum 12, allowing the investigation of proteins that can be modified at the N terminus or C terminus and are not secreted. A recent report describes protein mislocalisation induced by addition of a small molecule as a new conditional system to study protein functions in P. As a consequence, results from these studies can be difficult to interpret. Such systems allowing for control over mRNA abundance and stability, translation, and protein turnover have been developed but these approaches often result in only partial depletion of the gene product 8, 9, 10, 11. The parasite is haploid for much of its life cycle, including during the asexual erythrocytic cycle, requiring conditional genetic modification systems to study essential gene functions. Recently, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) genome editing system has been successfully adapted for use in Plasmodium spp., revolutionising genetic engineering in these parasites 5, 6, 7. It encodes roughly 5400 genes, half of which have no assigned function, in part due to the limited sequence similarity with genes outside of the Plasmodium genus. In 2002, the genome sequence of the 3D7 line of P. The fast spread of drug resistance 2 and the limited efficacy of current vaccines 3 highlight the pressing need to identify new targets for therapeutic intervention. falciparum infections, in particular affecting children below the age of five in Africa 1. 99% of these deaths occur as a consequence of P. Malaria is one of the most significant infectious diseases of people in low income countries, with an estimated 212 million cases in 2015 causing around half a million deaths. Lastly, we introduce an improved treatment protocol that uses a lower rapamycin concentration and shorter treatment times, leading to loxP-guided recombination with close to 100% efficiency within the same replication cycle. Furthermore, the newly developed 3D7-derived parasite lines, constitutively and stably expressing DiCre, generated using this toolkit will prove useful for the analysis of gene products. falciparum lines in any genetic background. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. We describe novel CRISPR/Cas9 transfection plasmids and approaches for the speedy, stable and marker-free introduction of transgenes encoding the DiCre recombinase into genomic loci dispensable for blood stage development. Here we employ CRISPR/Cas9 to facilitate use of the dimerisable Cre-recombinase (DiCre) that is frequently used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin controlled manner. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp.