International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 155-166 http://medscipublisher.com/index.php/ijmms 158 Figure 2 Mechanisms of CRISPR-Cas9 mediated genome engineering (Adapted from Jiang and Doudna, 2017) Image caption: The diagram illustrates how synthesized single-guide RNA (sgRNA) or the CRISPR RNA (crRNA)-tracrRNA complex directs the Cas9 endonuclease to target and cleave arbitrary DNA sequences in the genome, resulting in double-strand breaks (DSBs). Subsequently, host-mediated DNA repair mechanisms repair these breaks. In the absence of a repair template, the error-prone non-homologous end joining (NHEJ) pathway can lead to random insertions or deletions, thereby disrupting gene function. In the presence of a homologous repair template, the high-fidelity homologous recombination repair (HDR) pathway is activated to accurately repair or modify the target gene. The diagram also shows how altering the nuclease active sites of Cas9 (e.g., the D10A mutation) can achieve DNA targeting without cleavage, as well as other applications such as gene regulation, epigenetic modification, and live-cell imaging. These mechanisms demonstrate the wide potential applications of the CRISPR-Cas9 system in gene editing and regulation (Adapted from Jiang and Doudna, 2017) 2) Multiplexing Capability: CRISPR/Cas9 can target multiple genes simultaneously by using multiple guide RNAs. This multiplexing capability is crucial for comprehensive genetic modifications required in xenotransplantation to knock out multiple immunogenic genes (Tanihara et al., 2021). 3)Precision: The precision of CRISPR/Cas9 in targeting specific DNA sequences reduces the risk of off-target effects, which are common in other gene-editing technologies. This precision is enhanced by the ability to design highly specific guide RNAs (Matson et al., 2019). 4) Cost-Effectiveness: The lower cost and ease of designing CRISPR/Cas9 components make it more accessible for research and clinical applications compared to other gene-editing methods (Ryczek et al., 2021). 3.3 Applications of CRISPR/Cas9 in medical research CRISPR/Cas9 has been extensively utilized in medical research for disease modeling and therapeutic interventions. For instance, it has been employed to edit patient-derived xenografts (PDXs) to study genetic dependencies and mechanisms of drug resistance in cancer (Hulton et al., 2020). This approach allows for rapid in vivo functional genomics without the need for in vitro culture, significantly enhancing the utility of PDXs as models of human cancer.
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