PDF(Pubmed)
Abstract：
As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.
摘要：
至于许多其他生物，CRISPR-Cas9介导的遗传修饰对于新孢子虫疫苗候选物和药物靶标的鉴定越来越重要。一种导致牛流产和狗神经肌肉疾病的牙尖丛寄生虫。用于产生缺乏毒力因子的敲除(KO)菌株的广泛使用的方法是将药物选择标记如突变的二氢叶酸还原酶-胸苷酸合成酶(mdhfr-ts)整合到靶基因中。从而阻止各自蛋白质的合成并介导对乙胺嘧啶的抗性。然而,CRISPR-Cas9诱变并非没有脱靶效应，这可能导致多个mdhfr-ts拷贝整合到基因组的其他位点。要确定N.caninum中集成的mdhfr-ts的数量，开发了双重定量TaqManPCR。为此,设计引物，从野生型（WT）寄生虫中扩增与单拷贝wtdhfrs-ts基因相对应的106bp片段，以及KO寄生虫中存在的突变的mdhfrs-ts赋予抗性，并与扩增诊断性NC5基因的引物同时使用。因此，在WT寄生虫中，dhfr-ts与NC5的比率应约为1，而在具有单一整合mdhrf-ts基因的KO寄生虫中，这一比例翻了一番，并且在多个集成事件的情况下甚至更高。该方法应用于新孢子菌KO菌株NcΔGRA7和NcΔROP40。对于NcΔGRA7，通过dhfr-ts定量确定的速殖子数量是通过NC5定量确定的速殖子数量的两倍，因此表明只有一个mdhfr-ts副本被集成。用NcΔROP40菌株获得的结果，然而,显示每个基因组的dhfr-ts拷贝数要高得多，表明在CRISPR-Cas9的基因编辑过程中，至少三个拷贝的选择性mdhfr-ts标记被整合到基因组DNA中。这种双重TaqMan-qPCR提供了一种可靠且易于使用的工具，用于评估WT犬毒株中CRISPR-Cas9介导的诱变。
