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[用Cas9向部位对捐赠者DNA进行修改,提高了MTC34进入CXCR4位点的效率]

M V Shepelev¹, D S Komkov^1,2, D S Golubev¹

¹Center of Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia.

Molekuliarnaia biologiia

|December 22, 2024

概括

提高CRISPR/Cas9基因编辑需要高效的内置. 捐赠者DNA上的全长Cas9向位点 (CTS) 增加了两倍的内置水平,而截断的CTS (tCTS) 则没有,尽管在体外切割.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
克里斯普尔/卡斯9系统

背景情况:

基因组编辑CRISPR/Cas9的临床应用需要高的敲进效率.
目前加强内置的策略包括修改捐赠者DNA以Cas9准点 (CTS) 或截断版本 (tCTS).
这些捐赠者DNA修改改善敲门率的确切机制尚不清楚.

研究的目的:

评估全长CTS和tCTS修改对特定遗传结构的敲进效率的影响.
用CTS和tCTS修改后通过Cas9进行体外分离的供体DNA.

主要方法:

这项研究利用CRISPR/Cas9系统将MTC34遗传结构插入CEM/R5T细胞的CXCR4位点.
用全长CTS或tCTS修改了捐赠体等离子体,并评估了它们对敲门效率的影响.
进行了体外试验测试,以确定Cas9对修饰后的供体DNA的裂变.

主要成果:

将全长的CTS引入捐赠体等离子体上,使敲入水平翻了一番,不管CTS的数量或位置如何.
使用tCTS进行的修改并没有显著改变敲进效率.
全长CTS和tCTS都被Cas9在体外有效地切割.

结论:

在捐赠者DNA上进行全长CTS修改是提高CRISPR/Cas9中介敲门效率的可行策略.
在经过测试的条件下,截断的CTS修改似乎没有改善敲进率.
需要进一步的体内研究来阐明CTS和tCTS在基因组编辑中的作用的精确机制.

关键词:

这就是CRISPR/Cas9的作用.在Cas9中,目标网站是Cas9.核运输 DNA 核运输 DNA 在T细胞,T细胞.捐赠者的DNA 捐赠者的DNA 基因组编辑基因组编辑敲进来 - 敲进来

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