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包装的CRISPR-Cas9核糖蛋白加速了基因组编辑

Hannah Karp^1,2, Madeline Zoltek³, Kevin Wasko^2,3

¹Department of Chemistry, University of California, Berkeley, California 94720, USA.

bioRxiv : the preprint server for biology

|October 28, 2024

概括

通过包裹式输送车辆 (EDV) 输送CRISPR-Cas9核核蛋白 (RNP) 的效率是电穿孔的30倍以上. 这种包装的输送方法显著降低了有效的基因组编辑所需的剂量.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
基因编辑技术的技术

背景情况:

使用CRISPR-Cas9核糖蛋白 (RNP) 进行有效的基因组编辑,需要有效的细胞传递,同时尽量减少不良影响.
目前的临床应用主要使用电穿孔来进行ex vivo细胞修饰.
电穿孔和包装RNP交付方法之间缺乏系统的比较.

研究的目的:

为了比较两个CRISPR-Cas9 RNP输送策略的效率和剂量要求:电穿孔和封装输送车辆 (EDV).
确定生产性基因组编辑所需的Cas9 RNP最小剂量.
调查交付方法对RNP核停留时间和编辑动态的影响.

主要方法:

使用光相关谱法 (FCS) 来量化细胞内Cas9 RNP度.
在电穿孔和EDV输送系统之间比较基因组编辑效率和速度.
评估Cas9 RNP剂量要求,用于生产性编辑.

主要成果:

对于有效的基因组编辑,每个核通常需要超过1300个Cas9 RNP.
与电穿孔相比,EDV介导的交付效率提高了30倍以上.
基因组编辑发生的速度至少是EDV的两倍,RNP总剂量相比较.

结论:

封装式输送车辆 (EDV) 为CRISPR-Cas9 RNP输送提供了一种比电穿孔更有效的方法.
包装的RNP传递策略,如EDVs,可以大幅减少基因组编辑所需的CRISPR-Cas9 RNP的数量.
在EDV输送后增加RNP核停留时间可能有助于提高其有效性.

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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