TL;DR
This work describes how to generate degron knock-ins by CRISPR, cross them to TIR1-expressing strains, and perform depletion experiments, and provides a description of the current methods and highlight alternative approaches.
This protocol covers both single-gRNA (sgRNA) and dual-gRNA directed knockout systems and describes the procedure for constructing an Auxin-Inducible Degron (AID) knock-in-mediated system to induce the rapid degradation of a target protein in cells, thereby investigating its function.
Summary p53, a key tumor suppressor in animals, prevents DNA damage by arresting the cell cycle, activating repair, or inducing cell death. Mutations in p53 are found in over half of human cancers, making it vital for cancer research. To expand p53 research, we engineered Arabidopsis thaliana to express p53 under an inducible promoter, allowing precise control of its activity. The plant-produced p53 shares certain characteristics with mammalian p53 in molecular traits and subcellular localization. Interestingly, its overexpression triggers cell-cycle arrest followed by a developmental programmed cell death-like response. Copper ions also suppress p53 activity in this system. This tool also enables investigation of tissue regeneration using cell type-specific promoters, offering insights into p53’s broader roles and the regulation of cell death in plants.
A new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, such as clinical isolates and the reference strain SC5314 are reported, and it is demonstrated that the AID2 system also works in Candida auris, albeit less effectively under some conditions.
Caenorhabditis elegans (C. elegans) is a genetically tractable, transparent, and cost-effective metazoan model that bridges conventional cell-culture assays and mammalian systems for mechanistic cancer research. This pathway-focused review covers representative studies of conserved RAS/MAPK, Notch, and Wnt signaling and is organized around pathway conservation, mutant strains, quantitative phenotypes, genetic tools, and applications in mechanism studies, compound screening, and chemosensation-based assays. We first describe the conservation of pathway components and tumor-relevant phenotypes, including germline overproliferation and the multivulva (Muv) phenotype. We then catalog representative mutants, such as gain-of-function glp-1 and let-60 strains and loss-of-function gld-1 and lip-1 models, that enable analysis of stem-cell maintenance, ectopic proliferation, and signaling dysregulation. In addition, we discuss available tools for model construction, including RNA interference, transgenesis, MosTIC, TALENs, and CRISPR/Cas9, highlighting their strengths and limitations for cancer-related gene-function studies. Finally, we summarize published applications of C. elegans in anticancer drug screening, pathway discovery, and tumor-associated chemosensation. By integrating genetic conservation with scalable in vivo phenotyping, C. elegans provides a complementary platform for hypothesis generation, pathway validation, and early-stage therapeutic discovery, while its limitations in adaptive immunity and organ complexity should be recognized when translating findings to human cancer biology.
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