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文库筛选技术的发展及在肿瘤研究中的应用(4)
作者:网站采编关键词:
摘要:4 总结与展望 CRISPR/Cas9技术作为一个强大的基因编辑工具,凭借其制作简单、成本低、作用高效等优点广泛应用于细胞基因编辑和基因表达调控、基因敲除动
4 总结与展望
CRISPR/Cas9技术作为一个强大的基因编辑工具,凭借其制作简单、成本低、作用高效等优点广泛应用于细胞基因编辑和基因表达调控、基因敲除动物模型的构建、人类疾病动物模型的治疗研究等领域。而随着高通量文库筛选技术的建立,CRISPR/Cas9技术为研究肿瘤发生发展机制、研发抗癌药物以及肿瘤治疗等难题都提供了较好的解决方案。虽然CRISPR/Cas9技术存在较多优势,但高脱靶率、PAM 序列的限制、转导效率低等局限性仍限制着它的应用前景。因此,对于CRISPR/Cas9技术的运用主要应通过建立相应的人源化小鼠模型,以模拟人体疾病的发生和发展,并且在此基础上探索和研发有效的基因治疗手段,从而为相关疾病的后续药物开发和治疗提供重要的治疗方案和思路。当前来看,将基因编辑工具应用于人体之前必须有更低的CRISPR脱靶率和更先进的检测技术支持,以非常严谨的态度评估其中潜藏的各种风险,切不可急功近利。
基于CRISPR/Cas9技术的高通量文库筛选策略涉及多个过程,包括sgRNA的设计及合成、Cas9细胞系的构建、表型选择、高通量测序、生物信息学分析、候选基因和脱靶效应的验证等。CRISPR文库的构建流程和筛选效果方面,至少有3个方面可以进行提高:sgRNA的设计;sgRNA数量的优化;选择更合适的单克隆Cas9蛋白表达细胞。CRISPR/Cas9高通量筛选文库在哺乳动物细胞中的应用毫无疑问会为肿瘤等相关疾病机制的研究提供极大的帮助,我们可以预见,随着相关基础研究的深入,CRISPR/Cas9系统将在肿瘤个体化治疗方面得到更广阔的发展与应用。
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