Inducible high-efficiency CRISPR-Cas9-targeted gene editing and precision base editing in African trypanosomes

Eva Rico Vidal, Laura Jeacock, Julie Kovarova, David Horn (Lead / Corresponding author)

Research output: Contribution to journalArticle

10 Citations (Scopus)
142 Downloads (Pure)

Abstract

The Cas9 endonuclease can be programmed by guide RNA to introduce sequence-specific breaks in genomic DNA. Thus, Cas9-based approaches present a range of novel options for genome manipulation and precision editing. African trypanosomes are parasites that cause lethal human and animal diseases. They also serve as models for studies on eukaryotic biology, including 'divergent' biology. Genome modification, exploiting the native homologous recombination machinery, has been important for studies on trypanosomes but often requires multiple rounds of transfection using selectable markers that integrate at low efficiency. We report a system for delivering tetracycline inducible Cas9 and guide RNA to Trypanosoma brucei. In these cells, targeted DNA cleavage and gene disruption can be achieved at close to 100% efficiency without further selection. Disruption of aquaglyceroporin (AQP2) or amino acid transporter genes confers resistance to the clinical drugs pentamidine or eflornithine, respectively, providing simple and robust assays for editing efficiency. We also use the new system for homology-directed, precision base editing; a single-stranded oligodeoxyribonucleotide repair template was delivered to introduce a single AQP2 - T 791G/L 264R mutation in this case. The technology we describe now enables a range of novel programmed genome-editing approaches in T. brucei that would benefit from temporal control, high-efficiency and precision.

Original languageEnglish
Article number7960
Pages (from-to)1-10
Number of pages10
JournalScientific Reports
Volume8
Issue number1
DOIs
Publication statusPublished - 21 May 2018

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