Gene editing in Plasmodium berghei made easy: Development of a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation

Abstract

Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600-700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.

Keywords: CRISPR/Cas9; Gene targeting; Linear HDR; Plasmid; Plasmodium; Ribozyme; Transfection.