A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi.

VILCHEZ LARREA, SALOMÉ C.; PREGO, ALEJO; SCHOIJET, ALEJANDRA C.; LLANOS, MANUEL A.; ALBERCA, LUCAS N.; BELLERA, CAROLINA L.; GAVENET, LUCIANA; TALEVI, ALAN; ALONSO, GUILLERMO D.

Mendoza

Congreso; XI Congreso de la Sociedad Argentina de Protozoología; 2022

Sociedad Argentina de Protozoología

Development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to thestudy of Trypanosoma cruzi, an organism usually reluctant to other gene editing technologies.Most often, epimastigotes are co-transfected with a single plasmid bearing both the gene forCas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointlywith a lineal donor DNA encompassing a selection marker flanked by sequences homologous tothe target gene. Here, we tested an alternative approach for the generation ofPhosphodiesterase (PDE) knockout parasites. We obtained epimastigotes from Tul II strainstably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects onepimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to theintended gene target. sgRNA were obtained by in vitro transcription using a template DNAbearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA wedesigned a “pre-donor” formed by a sequence including several restriction enzyme recognitionsites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This“pre-donor” allowed to easily generate a variety of donor DNAs by cloning alternative selectionmarkers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated byPCR using “mixed” primer pairs: while one of the primers annealed to the target gene, thesecond primer annealed to a sequence in the donor DNA, allowing assessment of its correctinsertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include itsversatility for choosing and switching between alternative selection markers and a quick andaffordable generation of the components of the system and analysis of the transfected cultures,while possibly facilitating complementation assays on the KO lines.