QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii.
QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii.
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DOI：
10.3791/55185
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发表时间：
2017-06-22
期刊：
影响因子：
--
通讯作者：
Behnke MS
中科院分区：
文献类型：
--
作者：
Shen B;Powell RH;Behnke MS
科学知识与现有的技术和方法有着内在的联系。本文将介绍两种鉴定和验证顶复体寄生虫刚地弓形虫耐药基因的方法，一种是基于全基因组序列（WGS）遗传图谱的数量性状位点（QTL）定位方法，另一种是基于CRISPR /Cas9的聚类规则间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats， CRISPR）基因编辑方法。QTL定位的方法允许人们测试基因组区域和表型之间是否存在相关性。QTL扫描需要两个数据集，一个是基于重组杂交后代的遗传图谱，另一个是对该杂交后代的可量化表型进行评估。然后对这些数据集进行格式化，使其与R/qtl软件兼容，该软件生成qtl扫描，以识别与表型相关的重要位点。虽然这可以极大地缩小候选基因的搜索窗口，但qtl跨越了包含许多基因的区域，需要从中识别因果基因。对后代进行WGS检测对于在基因水平上确定耐药性突变的原因至关重要。一旦确定，候选突变可以通过药物敏感寄生虫的遗传操作来验证。对弓形虫进行基因改造最简单有效的方法是CRISPR/Cas9系统。该系统仅由两个组成部分组成，它们都编码在一个质粒上，一个是含有与基因组靶标互补的20 bp序列的单个引导RNA (gRNA)，另一个是在靶标上产生双链DNA断裂（DSB）的Cas9内切酶，其修复允许在断裂位点周围插入或删除序列。本文提供了使用基于CRISPR/Cas9的基因组编辑工具验证sinfungin抗性基因和构建转基因寄生虫的详细方案。
Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.
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