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Coupling chemical mutagenesis to next generation sequencing for the identification of drug resistance mutations in Leishmania

Nat Commun.. 2019; 
Bhattacharya A1, Leprohon P1, Bigot S1,2, Padmanabhan PK1, Mukherjee A1, Roy G1, Gingras H1, Mestdagh A1, Papadopoulou B1,2, Ouellette M3,4.
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摘要

Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin res... More

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