Efficient generation of a single-copy eft-3p::TIR1::F2A:: BFP::AID*::NLS allele in the C. elegans ttTi5605 insertion site through recombination-mediated cassette exchange.
Efficient generation of a single-copy eft-3p::TIR1::F2A:: BFP::AID*::NLS allele in the C. elegans ttTi5605 insertion site through recombination-mediated cassette exchange.
复制标题
DOI：
10.17912/micropub.biology.000425
复制
发表时间：
2021
影响因子：
--
通讯作者：
Ward JD
中科院分区：
文献类型：
--
作者：
Vo AA;Levenson MT;Ragle JM;Ward JD
生长素诱导降解系统(AID)是一种被广泛使用的条件性耗尽蛋白质的系统。利用CRISPR/Cas9在线虫中的基因组编辑，我们最近产生了一组单拷贝、组织特异性和泛体TIR1表达的菌株，携带一个BFP报告以单拷贝插入到两个常用的、特征良好的遗传位点。然而，我们未能获得一株携带泛体eft-3p：：TIR1：：F2a：：bfp：：aid*：：NLS转基因插入到染色体II ttTi5605插入位点的菌株。利用重组介导盒交换(RMCE)，我们能够有效地获得这种敲入。与我们之前产生的进入染色体I ttTi4348插入位点的转基因eft-3p：：TIR1：：F2a：：BFP：：Aid*：：NLS相比，得到的菌株显示出相当于AID*：：GFP报告基因的耗尽。这项工作突出了RMCE为AID系统产生新试剂的能力，并在第二染色体上提供了eft-3p：：TIR1：：F2a：：bfp：：aid*：：NLS等位基因，当使用AID系统时，它将简化遗传杂交方案。
The auxin-inducible degron (AID) system is a widely used system to conditionally deplete proteins. Using CRISPR/Cas9-based genome editing in C. elegans, we recently generated a set of single-copy, tissue-specific and pan-somatic TIR1-expressing strains carrying a BFP reporter inserted in single-copy into two commonly used, well-characterized genetic loci. However, we were unable to obtain a strain carrying a pan-somatic eft-3p::TIR1::F2A::BFP::AID*::NLS transgene inserted into the chromosome II ttTi5605 insertion site. Using recombination-mediated cassette exchange (RMCE) we were able to efficiently obtain this knock-in. The resulting strain displayed equivalent depletion of an AID*::GFP reporter compared to our previously generated eft-3p::TIR1::F2A::BFP::AID*::NLS transgene knocked into the chromosome I ttTi4348 insertion site. This work highlights the power of RMCE for generating new reagents for the AID system and provides an eft-3p::TIR1::F2A::BFP::AID*::NLS allele on chromosome II which will simplify genetic crossing schemes when using the AID system.