Streptococcus pyogenesCas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing inTrypanosomaandLeishmania

Abstract

SUMMARYKinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmaniaspp.), as well as several animal diseases with important economic impact (African trypanosomes, includingT. congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinantStreptococcus pyogenesCas9 (SpCas9) and anin vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete and mutate candidate genes in various stages of the life cycle ofT. bruceiandT. congolense, andLeishmaniapromastigotes. The functionality ofSpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics. Importantly, this approach is adaptable to any wild-type parasite, including field isolates.