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大基因在哺乳动物细胞中通过不断进化的重组酶和原始编辑进行高效的特定位点集成

Smriti Pandey^1,2,3, Xin D Gao^1,2,3, Nicholas A Krasnow^1,2,3

¹Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Nature biomedical engineering

|June 10, 2024

概括

菌体辅助的连续进化显著增强了主要编辑辅助的特定位点整合酶基因编辑 (PASSIGE). 这种新的方法在哺乳动物细胞中实现了高效,大规模的DNA载荷集成,克服了以前的局限性.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
基因组学就是基因组学.

背景情况:

哺乳动物基因组中现有的基因整合方法面临着可编程性,效率和特异性的挑战.
精确整合大型DNA序列 (超过10kb) 仍然是基因工程中的一个重大障碍.

研究的目的:

通过菌体辅助的连续进化来增强主要编辑辅助的特定位点整合酶基因编辑 (PASSIGE).
开发一种高效和可编程的方法,以有针对性地将大型DNA载荷集成到哺乳动物基因组中.

主要方法:

使用菌体辅助的连续进化来设计Bxb1重组酶变体 (evoBxb1和 eeBxb1).
结合主要编辑可编程性与工程重组酶特异性,用于特定站点的DNA集成.
在人类细胞系中评估了整合效率,包括安全港和治疗相关地点,以及人类原发性纤维细胞.

主要成果:

工程重组酶变体 (evoBxb1,eeBxb1) 实现了高达60%的供体集成,比野生型Bxb1.1增加了3.2倍.
在单个转染实验中,PASSIGE与eeBxb1的平均向基因整合效率为23%,比野生类型高4.2倍.
在人类原发性纤维细胞中,整合效率超过了30%,明显超过PASTE方法.

结论:

PASSIGE,特别是随着不断进化的重组酶,代表了在哺乳动物细胞中向基因整合的高效战略.
这种方法克服了以前方法的局限性,使大DNA有效载荷的精确集成成为可能.
开发的方法有望促进基因疗法和合成生物学应用.

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