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在基于CRISPR的转录激活系统中激活剂表达的细胞毒性

Aakaanksha Maddineni¹, Ziyan Liang¹, Shreya Jambardi¹

¹Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA.

bioRxiv : the preprint server for biology

|October 10, 2024

概括

常见的基于CRISPR的转录激活系统 (CRISPRa) 显示出显著的细胞毒性. 这种与强有力的激活剂相关的细胞毒性限制了CRISPRa.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
细胞毒理学细胞毒理学

背景情况:

基于CRISPR的转录激活 (CRISPRa) 是调节基因表达的强大工具.
现有的CRISPRa系统,特别是协同激活调解器 (SAM) 系统,在研究中得到广泛使用.
关于CRISPRa工具的安全性和可靠性的担忧正在出现.

研究的目的:

为了研究常用的CRISPRa系统的细胞毒性.
评估表达转录激活域对细胞活性的影响.
确定当前广泛应用的CRISPRa技术的潜在局限性.

主要方法:

表达p65和HSF1激活域 (ADs) 的CRISPRa载体的评估毒性.
将研究扩展到其他ADS和p300乙转移酶核心领域.
使用可诱导的lentiviral向量来探索激活器表达窗口.

主要成果:

已发表的和新的CRISPRa载体显示出显著的细胞毒性.
强大的转录激活剂的表达降低了lentiviral标位,并导致细胞死亡.
在没有可测量的毒性的情况下,没有确定基于SAM的CRISPRa的安全窗口.

结论:

包括SAM在内的当前CRISPRa系统的细胞毒性阻碍了它们的广泛使用.
毒性引入了选择瓶,可能会损害基因查结果.
未来的CRISPR开发必须平衡激活效率与降低激活剂毒性.

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