CRISPRing Toxoplasma gondii: our experience

LAURA VANAGAS; DANIELA MUÑOZ; CONSTANZA CRISTALDI; SERGIO O. ANGEL

Mar del Plata

Congreso; Reunión anual de sociedades de Biociencia SAIC.SAFE.SAB.SAP. AACYTAL.NANOMED-ar.HCS; 2019

Nowadays CRISPR (clusteredregularly interspaced short palindromic repeats) system, a naturally occurringadaptive immune system found in bacteria and archaea, is widely adapted fortargeted genome editing in a variety of organisms, and Toxoplasma gondii is not the exception. In 2014, two groupsindependently adapted in a single plasmid the CRISPR/Cas9 system to express in T.gondii both the guide RNA and the Cas9nuclease providing an efficient and powerful tool to the community for testingthe role of specific genes in diverse genetic backgrounds of T. gondii. Since then many improvementshave been incorporated, making this technology available not only for thisparasite, but also for closely related ones, such as Neospora caninum. Regarding T.gondii,this technology has made it possible to perform a genome-wide genetic screen whichhas provided all the community with the great possibility to have an idea onthe contributions to parasite fitness of every protein prior to starting astudy. This work provided us with the explanation to our impossibility toobtain a knock-out of both H2A.Z and H2B.Z histones, which were shown to beprobably essential. In this context, we have been successful in using CRISPR/Cas9to genetically manipulate T. gondiilines that had been obtained earlier, in which H2B.Z was over-expressed andtagged with c-Myc, with different mutations, in order to eliminate theendogenous protein. We are also actually working on the generation of aknock-out of H2A.X histone, which should not be essential for the parasitesurvival, according to the above mentioned study. The incorporation of thisuseful tool has been important for our lab, providing a more accessible way togenetically manipulate the parasite and we intend to use it not only to knockproteins out, but also for allelic replacements or mutagenesis.