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毛针内部核定位信号在CRISPR-Cas9中增强了初级人类淋巴细胞的编辑

Eric A Noel^1,2,3, Srishti U Sahu^1,2,3, Stacia K Wyman¹

¹Innovative Genomics Institute, University of California Berkeley, Berkeley, California, USA.

The CRISPR journal

|March 31, 2025

概括

内部核定位信号 (NLS) 序列提高了CRISPR-Cas9编辑效率和人类T细胞中的蛋白质产量. 这种新的内部NLS (hiNLS) 策略为研究和治疗提供了增强的基因组编辑结果.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
细胞工程细胞工程

背景情况:

核定位信号 (NLS) 序列对于将CRISPR酶引导到细胞核中进行基因组编辑至关重要.
终端NLS融合增强了CRISPR酶的性能,但可以减少重组表达期间的蛋白质产量.
优化CRISPR酶的递送和效率仍然是基因编辑应用中的一个关键挑战.

研究的目的:

开发一种新的战略,以提高CRISPR-Cas9基因组编辑效率和蛋白质生产.
为了研究头内部NLS (hiNLS) 序列在CRISPR-Cas9骨干中集成的疗效.
为了比较hiNLS Cas9变体与人类T细胞中终端融合的NLS结构的性能.

主要方法:

设计和制造了具有发针内部NLS (hiNLS) 序列的工程CRISPR-Cas9变体.
hiNLS Cas9 变种在人类初级T细胞中对基因编辑效率进行了评估.
通过电穿孔和与两性的共同化实现了核蛋白复合体的输送.

主要成果:

hiNLS Cas9变体在人类T细胞中表现出与终端NLS融合相比更好的基因编辑效率.
许多hiNLS Cas9构造表现出高纯度和产量,即使有多个NLS插入 (最多9个).
hiNLS策略成功地促进了核定位和高效的DNA编辑.

结论:

针头内部NLS (hiNLS) 序列代表了CRISPR效应器设计的重大进步.
这种方法克服了终端NLS融合的局限性,提高了编辑效率和蛋白质产量.
hiNLS Cas9构造对研究和治疗应用中增强基因组编辑结果具有前景.

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