Abstract
Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. 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. 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. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. 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. 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.
Highlights
This page was generated automatically upon download from the ETH Zurich Research Collection
We describe a toolkit of CRISPR/Cas[9] and rescue plasmids to enable the quick, stable and marker-free generation of dimerisable Cre-recombinase (DiCre)-expressing P. falciparum parasites. We demonstrate this by inserting DiCre recombinase into two loci, p230p and pfs[47], which are dispensable for blood stage development as well as for infectivity to mosquitoes
On the other hand after three months in culture, PCR amplification from the 1G5DC reporter line showed a band corresponding to an intact SERA5 locus, suggesting that over time the DiCre cassette is lost in part of the population. These results show that the new transgenic P. falciparum lines II-3 and Pfs[] stably maintain DiCre recombinase and excise loxP-flanked DNA sequences with extremely high efficiency even at a 10-fold lower rapamycin concentration than has been routinely used
Summary
This page was generated automatically upon download from the ETH Zurich Research Collection. We describe novel CRISPR/Cas[9] 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 Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. 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 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].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
