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

Eva Rico,Laura Jeacock,David Horn,Julie Kovářová

doi:10.1038/s41598-018-26303-w

Eva Rico, Laura Jeacock + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-26303-w

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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 - T791G/L264R 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.

Highlights

The African trypanosomes of the Trypanosoma brucei group are transmitted by tsetse flies and cause lethal diseases in humans and livestock, known as sleeping sickness and nagana, respectively
For T. brucei, double-allele editing has not been achieved without selectable markers nor have efficiencies been determined
The current strategy involves annealing a pair of oligonucleotides and ligating the product to the BbsI-digested pT7sgRNA construct prior to transfection

Summary

Introduction

The African trypanosomes of the Trypanosoma brucei group are transmitted by tsetse flies and cause lethal diseases in humans and livestock, known as sleeping sickness and nagana, respectively Research on these protozoan parasites, and related parasitic trypanosomatids, often focuses on virulence mechanisms and druggable biology[1]. Chromosomal site-specific DNA double strand breaks (DSBs) can increase recombination efficiency in T. brucei, as demonstrated following inducible expression of the I-SceI meganuclease[8]. In the bacterial cells, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), derived from invading phage DNA, produce CRISPR RNA (crRNA) which, together with a trans-activating crRNA (tracrRNA) program Cas[9] (CRISPR associated protein 9) to cut both the RNA-complementary and non-complementary strands, producing DSBs in the target DNA, 3 bp upstream of the PAM. The crRNA and tracrRNA can be fused to generate a chimeric single guide RNA (sgRNA)[11], such that a two-component system requires only Cas[9] and an sgRNA for programmed editing in a heterologous system

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