Genome deletions to overcome the directed loss of gene function in Leishmania.
Genome deletions to overcome the directed loss of gene function in Leishmania.
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DOI：
10.3389/fcimb.2022.988688
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发表时间：
2022
影响因子：
5.7
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中科院分区：
文献类型：
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随着被忽视的热带病利什曼病的全球影响范围不断扩大，加上治疗药物的数量很少，这些药物都存在耐药性、成本、毒性和/或给药方面的问题，验证由昆虫媒介传播的原生动物利什曼原虫的新药靶标比以往任何时候都更加重要。在2015年引入CRISPR Cas9技术之前，新靶点的遗传验证主要是通过同源重组进行靶向基因敲除，大多数靶向基因(~70%)被认为是非必需的。在这项研究中，我们利用现成的全基因组测序技术来重新分析这些历史悠久的细胞系之一，丝氨酸棕榈酰转移酶(LCB2)催化亚基中的L.主要基因敲除，它导致鞘磷脂生物合成的完全丧失，但仍具有活性和感染性。这揭示了一些单核苷酸多态，但也完全丢失了几个编码区，包括一个编码推测的ABC3A同源基因的基因，一个推测的固醇转运蛋白。假设失去这样的转运蛋白可能促进了LCB2催化亚单位的定向敲除和完全丧失了从头开始的鞘脂生物合成，我们重新检查了一株为直接CRISPR Cas9定向操作而设计的墨西哥乳杆菌系中的LCB2。令人惊讶的是，LCB2不能被敲除，这表明了重要性。然而，同时缺失LCB2和推测的ABC3A是可能的。这表明推测的ABC3A的缺失促进了利什曼原虫神经鞘脂脂生物合成的丧失，并建议我们应该重新检查许多其他利什曼原虫基因被认为是非必需的敲除株系。
With the global reach of the Neglected Tropical Disease leishmaniasis increasing, coupled with a tiny armory of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyze one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesizing that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential.