Khan Haania, Huang Xinyu, Raj Vishnu, Wang Han
Department of Integrative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS Genet. 2025 Jan 22;21(1):e1011541. doi: 10.1371/journal.pgen.1011541. eCollection 2025 Jan.
The ability to manipulate gene activity and control transgene expression is essential to study gene function. While several genetic tools for modifying genes or controlling expression separately are available for Caenorhabditis elegans, there are no genetic approaches to generate mutations that simultaneously disrupt gene function and provide genetic access to the cells expressing the disrupted gene. To achieve this, we developed a versatile gene trap strategy based on cGAL, a GAL4-UAS bipartite expression system for C. elegans. We designed a cGAL gene trap cassette and used CRISPR/Cas9 to insert it into the target gene, creating a bicistronic operon that simultaneously expresses a truncated endogenous protein and the cGAL driver in the cells expressing the target gene. We demonstrate that our cGAL gene trap strategy robustly generated loss-of-function alleles. Combining the cGAL gene trap lines with different UAS effector strains allowed us to rescue the loss-of-function phenotype, observe the gene expression pattern, and manipulate cell activity spatiotemporally. We show that, by recombinase-mediated cassette exchange (RMCE) via microinjection or genetic crossing, the cGAL gene trap lines can be further engineered in vivo to easily swap cGAL with other bipartite expression systems' drivers, including QF/QF2, Tet-On/Tet-Off, and LexA, to generate new gene trap lines with different drivers at the same genomic locus. These drivers can be combined with their corresponding effectors for orthogonal transgenic control. Thus, our cGAL-based gene trap is versatile and represents a powerful genetic tool for gene function analysis in C. elegans, which will ultimately provide new insights into how genes in the genome control the biology of an organism.
操纵基因活性和控制转基因表达的能力对于研究基因功能至关重要。虽然有几种分别用于修饰基因或控制表达的遗传工具可用于秀丽隐杆线虫，但尚无遗传方法来产生同时破坏基因功能并提供对表达被破坏基因的细胞进行遗传访问的突变。为实现这一目标，我们基于cGAL开发了一种通用的基因捕获策略，cGAL是一种用于秀丽隐杆线虫的GAL4-UAS二元表达系统。我们设计了一个cGAL基因捕获盒，并使用CRISPR/Cas9将其插入目标基因，创建一个双顺反子操纵子，该操纵子在表达目标基因的细胞中同时表达截短的内源蛋白和cGAL驱动蛋白。我们证明我们的cGAL基因捕获策略能稳健地产生功能丧失等位基因。将cGAL基因捕获系与不同的UAS效应菌株相结合，使我们能够挽救功能丧失表型、观察基因表达模式并时空操纵细胞活性。我们表明，通过显微注射或遗传杂交的重组酶介导的盒式交换（RMCE），cGAL基因捕获系可在体内进一步工程化，以轻松地将cGAL与其他二元表达系统的驱动蛋白（包括QF/QF2、Tet-On/Tet-Off和LexA）进行交换，从而在同一基因组位点产生具有不同驱动蛋白的新基因捕获系。这些驱动蛋白可与其相应的效应器结合用于正交转基因控制。因此，我们基于cGAL的基因捕获具有通用性，是秀丽隐杆线虫基因功能分析的强大遗传工具，最终将为基因组中的基因如何控制生物体生物学提供新的见解。
