Cancer Genetics and Epigenetics 2024, Vol.12, No.5, 294-305 http://medscipublisher.com/index.php/cge 299 can lead to imprecise insertions or deletions. To address these issues, researchers are exploring alternative repair mechanisms such as microhomology-mediated end joining (MMEJ). When using CRISPR-Cas9 technology to inject specific sgRNAs into zebrafish embryos, phenotypes such as mild cardiac edema were observed. The experiments demonstrated good reproducibility and high mutation efficiency. This mechanism shows potential in reducing unwanted mutations by promoting more predictable outcomes (Ata et al., 2018) (Figure 2). Despite advances, fine-tuning gene editing tools to achieve cancer-specific precision remains a significant hurdle. Figure 1 ID8 retains wild-type p53 function and demonstrates competent homologous recombination (Adopted from Walton et al., 2016) Note: A: expression of p53 was assessed in ID8 cells following treatment for up to 8 hours with 10 μmol/L cisplatin (left) or 10 μ mol/L Nutlin-3 (right). B: transcription of p53 target Cdkn1a in ID8 cells following treatment for up to 8 hours with 10 μmol/L cisplatin was assessed by quantitative reverse-transcriptase PCR, normalized to Rpl34. Data represent mean ± SD (n = 3) plotted relative to untreated cells (t = 0 hours). C: representative chromatograms of Sanger sequencing of p53 from parental ID8 tumors harvested after 10 days intraperitoneal growth in female C57Bl/6 mice, demonstrating no mutations at critical hotspot mutations sites at residues 172, 217, 245, and 270—the equivalent human codons are shown in parentheses below. D: ID8 tumors, fixed in 4% neutral-buffered formalin, were stained for p53, WT1, and PAX8. Bars, 100 μm. E: ID8 cells were irradiated (10 Gy) or treated with rucaparib (10 μmol/L for 24 hours), fixed and stained for γH2AX and RAD51, and counterstained with DAPI. RAD51 foci were counted in up to 30 untreated and rucaparib-treated cells. Bars, mean (± SD) RAD51 foci per cell; dotted line, two-fold increase in RAD51 foci/cell relative to untreated cells, suggestive of functional homologous recombination (21) (Adopted from Walton et al., 2016).
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