Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker
Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker
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DOI：
10.1128/ec.00229-14
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发表时间：
2015-02-01
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影响因子：
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通讯作者：
Sibley, L. David
中科院分区：
文献类型：
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作者：
Behnke, Michael S.;Khan, Asis;Sibley, L. David
数量性状基因座（QTL）定位研究已在确定和了解寄生虫弓形虫的毒力机制不可或缺。在这项研究中，我们通过在2型ME 49-FUDRr和10型VAND-SNFr之间的遗传杂交中定位sinefungin（SNF）抗性来询问不同的表型。通过对后代进行全基因组测序并随后鉴定从父母遗传的单核苷酸多态性（SNP）来生成该杂交的遗传图谱。基于这个高密度的遗传图谱，我们能够将sinefungin抗性表型精确定位到染色体IX上的一个重要位点。在该基因座内，鉴定了导致TGVAND_290860基因中的早期终止密码子的单个非同义SNP（nsSNP），其仅发生在耐sinefungin的后代中。使用CRISPR/CAS9，我们能够证实TGVAND_290860的靶向破坏使寄生虫具有sinefungin抗性。由于SNR 1基因的破坏赋予耐药性，我们还表明，它可以被用作一个负选择标记插入一个积极的药物选择盒或异源报告。这些数据证明了将经典遗传作图、全基因组测序和CRISPR介导的基因破坏相结合，用于T.刚地。
Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDRr and type 10 VAND-SNFr. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.