Transfection of P. knowlesi in vitro and P. cynomolgi in vivo using the CRISPR/Cas9 technology

Abstract

Genomic editing techniques are facilitating the development of transgenic organisms, increasing efficiency and reducing the time required for the selection of these lines. Among these techniques is CRISPR/Cas9, which is based on the repair of DNA double strand breaks (DSBs) induced at specific sites in the genome. After DSBs the cells quickly repair the DNA, using homologous (HR) or non-homologous recombination pathways. During HR repair the cell machinery uses a sequence with homologous ends to the target sequence, which may contain mutations or insertions of new genes. Recently, the group of Dr. Robert Moon, from the United Kingdom, adapted the CRISPR/Cas9 technology to P. knowlesi allowing fast generation of transgenic parasites for studies of different characteristics.In this project, we will estabilish this technology in our laboratory, generating transgenic P. knowlesi lines and adapt the technology to P. cynomolgi - species that never had its DNA modified by genomic editing techniques. We contacted Dr. Moon's group, which has already provided us the plasmids and protocols to establish the technique, also offering support. We will generate a transgenic reporter line containing the bioluminescent protein NanoLuc gene, inserted in the P. knowlesi genome. For that, we will insert a "cassette" containing the pkhsp70 gene promoter sequence, driving NanoLuc expression, in the locus of the p230p gene, previously altered by Dr. Moon's group - which didn't result in functional changes. The transgenic bioluminescent parasites will be used in drug activity and mosquito transmission assays, which will be done in partnership with the group of Dr. Thomas Wellems (NIH, Bethesda-USA). The p230p gene will also be used as target for genomic editing of P. cynomolgi, obtained in experimental infections of rhesus and cynomolgous monkeys, which will occur at the ICTB (Institute of Science and Technology of Biomodels - FIOCRUZ), in collaboration with the group of Dr. Leonardo Carvalho (FIOCRUZ - RJ).We will also generate gametocyte reporter lines, to isolate these evolutionary forms. For this goal, we will insert in the p230p locus a "cassette" containing the promoter of a specific gametocyte gene (pks16) driving the expression of the fluorescent protein mCherry. Only the gametocytes will become red, allowing their isolation by flow cytometry. These parasites will allow us to study details of these evolutionary forms, which occur in low numbers in P. knowlesi and are essential for mosquito transmission. (AU)