International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 155-166 http://medscipublisher.com/index.php/ijmms 159 The study by Hulton et al. (2020) explored a method for rapid in vivo functional genomics research by directly editing the genomes of patient-derived xenografts (PDXs) using CRISPR-Cas9. They developed a technique that employs a tightly regulated, inducible Cas9 vector, which allows for the selection of transduced cells without the need for in vitro culture. This approach enabled the analysis of genetic dependencies in PDXs through targeted gene disruption and the investigation of acquired drug resistance mechanisms via homology-directed repair (HDR). The results demonstrated that this versatile system could be broadly applied to other xenograft models, significantly enhancing the utility of PDXs as genetically programmable models for human cancer. This method greatly improves the value of PDX models in cancer biology research and the development of new therapeutic strategies. Figure 3 Design of the pSpCTRE vector and its validation in vitro (Adapted from Hulton et al., 2020) Image caption: a shows the structure of the pSpCTRE vector, which includes a TRE3GS promoter for doxycycline (dox)-induced expression of Cas9 and a CD4T cell surface marker to facilitate rapid selection of transduced cells; b demonstrates the results of CD4T selection and enrichment of A549 cells using flow cytometry; c and d validate the efficient expression of Cas9 and the effective editing of the GFP gene in the presence of dox, as well as the absence of Cas9 expression and GFP editing without dox, through a GFP editing experiment; e displays the dose-dependent effect of different dox concentrations on Cas9 expression and GFP editing efficiency; f shows that A549GFP-SpCTRE cells passaged multiple times do not exhibit leaky expression of Cas9 in the absence of dox, while effective GFP editing occurs upon dox addition (Adapted from Hulton et al., 2020) Hulton et al. (2020) discovered that by integrating the tightly regulated TRE3GS promoter and the CD4T cell surface marker, the pSpCTRE vector can rapidly select and enrich transduced cells in A549 cells (Figure 3a and 3b). Under tetracycline (dox) induction, the pSpCTRE vector efficiently expresses Cas9 and successfully edits the GFP gene, whereas in the absence of dox, Cas9 is not expressed and GFP remains unedited (Figure 3c and 3d). This indicates that the pSpCTRE system has low background activity without the inducer. The dose-dependent response of Cas9 expression and GFP editing to dox concentration demonstrates the vector system’s sensitive response to dox (Figure 3e). Even after multiple passages, A549GFP-SpCTRE cells remain stable under no-dox conditions and can perform effective editing immediately upon dox addition (Figure 3f). These results suggest that the pSpCTRE vector exhibits high controllability and efficacy in vitro, laying the foundation for further in vivo functional genomics research. Furthermore, CRISPR/Cas9 has been used to investigate gene editing in human tripronuclear zygotes, revealing insights into DNA repair mechanisms and the challenges of off-target effects and mosaicism in early embryos
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