CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi

Abstract

Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi, demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome.

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor

“…Trypanosoma cruzi, the etiologic agent of Chagas disease, which is a significant cause of morbidity and mortality from the South of the United States to the South of Argentina and Chile, has been particularly refractory to genetic manipulation. However, the recent use of the CRISPR/Cas9 technology to knock down or knock out genes (2,3) has revolutionized their study.…”

Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominanttrans-Sialidase-Specific CD8+T Cells

“…A test of this hypothesis will require ablating the TS genes from T. cruzi's genome, a challenging endeavor given the thousands of TS genes. However, recent developments in the clustered regularly interspaced short palindromic repeat(s) (CRISPR)/Cas9 genome-editing technology may provide us with this opportunity in the near future (63).…”

Current and Future Chemotherapy for Chagas Disease

“…Most recently, and in the wake of a revolution in genome editing technology, CRISPR-Cas9 technology has been successfully applied to T. cruzi with major breakthroughs like expression knocking down of an enzyme gene family consisting of 65 members 44 .…”