CRISPR-mediated Transfection of Brugia malayi
CRISPR-mediated Transfection of Brugia malayi
复制标题
DOI：
10.1371/journal.pntd.0008627
复制
发表时间：
2020-08-01
影响因子：
3.8
通讯作者：
Unnasch, Thomas R.
中科院分区：
文献类型：
--
作者：
Liu, Canhui;Grote, Alexandra;Unnasch, Thomas R.
反向遗传学在人类丝虫寄生虫中的应用由于这些生物体的生物学困难而滞后。最近，我们开发了一种共培养系统，使感染性马来丝虫幼虫阶段被转染并有效地发育为有生殖力的成虫。这被用来开发基于apiggyBactransposon的工具包，该工具包可用于生产具有稳定整合到寄生虫基因组中的转基因序列的寄生虫。然而，piggyBac系统通常被基于CRISPR的技术所取代，该技术允许精确编辑基因组。在这里，我们报告了将piggyBac-mediated转染系统用于马来丝虫的CRISPR介导的敲入插入寄生虫基因组。在马来双歧杆菌基因组的基因间区域中鉴定了合适的CRISPR插入位点。修饰了一个双链piggybac载体，用插入位点侧翼的序列取代piggybac反向末端重复序列。用合成的指导RNA、修饰的质粒和CAS 9核酸酶转染马来酸B.malayimolting L3。将转染的寄生虫植入沙鼠中并使其发育成成虫。回收子代微丝蚴并筛选质粒中编码的分泌型荧光素酶报告基因的表达。发现大约3%的微丝蚴分泌荧光素酶;所有微丝蚴都含有插入寄生虫基因组中预期位置的转基因序列。使用衔接子介导的PCR检测，检测转基因微丝蚴是否存在脱靶插入;没有发现脱靶插入。这些数据证明CRISPR可用于修饰马来双歧杆菌的基因组，为精确编辑这种重要的人类丝虫寄生虫的基因组开辟了道路。作者摘要人类丝虫寄生虫是淋巴丝虫病的病原体象皮病（象皮病）和盘尾丝虫病（河盲症）是世界范围内发病的一些最重要原因。对这些生物体的研究的一大障碍是无法采用反向遗传方法和开发整合的转基因寄生虫系。最近，我们开发了一种基于apiggyBac转座子的方法，该方法采用共培养系统，允许在培养物中通过脂质体转染感染性幼虫阶段的马来丝虫，从而产生发育能力的转基因寄生虫。然而，piggyBac系统不能用于精确编辑基因组中的特定序列。因此，piggyBacsystem通常被基于CRISPR的技术所取代，该技术允许精确靶向（和编辑）基因组中的特定序列。在这里，我们报告了在为piggyBac介导的马来丝虫转染开发的方法的基础上，开发了一种CRISPR介导的方法，用于在这种寄生虫中进行精确的转基因。
The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage ofBrugia malayito be transfected and efficiently develop to fecund adults. This was exploited to develop apiggyBactransposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, thepiggyBacsystem has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting thepiggyBacmediated transfection system ofB.malayifor CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of theB.malayigenome. A dual reporterpiggybacvector was modified, replacing thepiggyBacinverted terminal repeat regions with sequences flanking the insertion site.B.malayimolting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome ofB.malayi, opening the way to precisely edit the genome of this important human filarial parasite.Author summary Human filarial parasites are the causative agents of lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness) and are some of the most important causes of morbidity worldwide. A large obstacle to research on these organisms has been the inability to employ reverse genetic methods and to develop integrated transgenic parasite lines. Recently, we developed apiggyBactransposon-based method that employed a co-culture system that permitted the infective larval stage ofB.malayito be transfected by lipofection in culture, resulting in the production of developmentally competent transgenic parasites. However, thepiggyBacsystem cannot be used to precisely edit particular sequences in the genome. Thus, thepiggyBacsystem has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which permits precise targeting (and editing) of particular sequences in the genome. Here, we report building upon the methods developed forpiggyBacmediated transfection ofB.malayito develop a CRISPR mediated method for precise transgenesis in this parasite.