Cancer Genetics and Epigenetics, 2025, Vol.13, No.1, 41-49 http://medscipublisher.com/index.php/cge 45 5 Limitations and Challenges of Current Breast Cancer Applications 5.1 Non-targeted risks of gene editing and genomic perturbation Although the CRISPR/Cas9 technology is innovative, there are still technical challenges in off-target editing. Unexpected genetic modifications may cause genomic instability, which is particularly crucial for the treatment of breast cancer. When the editing system acts on homologous sequence regions, unplanned genetic variations may occur, thereby accelerating tumor progression or triggering novel mutations (Wen and Zhang, 2022; Guo et al., 2023). Although the development of high-fidelity Cas9 variants has improved the targeting accuracy, off-target effects remain the core limitation hinders clinical application (Allemailem et al., 2023). The complexity of the genomic repair mechanism further increases the security risks. Crispr-induced DNA double-strand breaks may trigger large fragment deletions or chromosomal structural abnormalities, resulting in the loss of functional alleles. Eliminating these risks is the basis for the development of precision treatment, and there is an urgent need to develop an editing system with stronger predictability (Wen and Zhang, 2022; Guo et al., 2023). 5.2 Technical barriers to in vivo targeted delivery There are significant technical bottlenecks in achieving the specific delivery of CRISPR components in breast tumors. The ideal delivery system needs to accurately identify the lesion area and avoid the exposure of normal tissues. Existing viral vectors and non-viral vectors all face defects such as immune activation, load limitation and non-specific distribution (Figure 2) (Wei et al., 2020; Allemailem et al., 2022). Although the virus delivery system has a relatively high transfection efficiency, its immunogenicity and limited genetic material carrying capacity restrict its applicability in the treatment of breast cancer (Wei et al., 2020). Figure 2 Different strategies of CRISPR/Cas9 delivery as mRNA, DNA, or protein (Adopted from Allemailem et al., 2022) Image caption: These methods include adenovirus transport, electroporation, microinjection, and the use of multifunctional NPs; Abbreviations: Cas9/sgRNA, CRISPR associated protein 9/single-guide RNA; CRISPR, clustered regularly interspaced short palindromic sequence; NPs, nanoparticles; NPC, nuclear pore complex (Adopted from Allemailem et al., 2022)
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