具有广泛PAM兼容性和高DNA特异性的进化Cas9变体
Johnny H Hu1,2,3, Shannon M Miller1,2,3, Maarten H Geurts1,2,3
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
Nature
|March 8, 2018
概括
科学家开发了一种扩展的CRISPR-Cas9变体 (xCas9),可以识别更广泛的DNA序列. 这一突破提高了人类细胞的基因组编辑能力和特异性.
科学领域:
- 分子生物学
- 遗传学
- 生物技术
背景情况:
- CRISPR-Cas9基因组编辑依赖于识别特定的DNA序列,称为原空间器相邻动机 (PAM).
- 广泛使用的Streptococcus pyogenes Cas9 (SpCas9) 仅限于NGG PAM序列.
- 现有的Cas9变种为哺乳动物细胞应用提供有限的PAM灵活性.
研究的目的:
- 设计一个具有扩展PAM识别配置的Cas9变种.
- 评估工程变异在人类细胞中的功能性和特异性.
- 扩大CRISPR-Cas9技术的目标范围.
主要方法:
- 使用菌体辅助连续进化 (PACE) 进化了一个扩展的PAM SpCas9变体 (xCas9).
- 功能评估包括针对性的转录激活,基因破坏和人类细胞的基因编辑.
- 评估了全基因组的脱活动以确定DNA的特异性.
主要成果:
- 进化的xCas9变种识别了广泛的PAM序列,包括NG,GAA和GAT.
- 在哺乳动物细胞中活跃的Cas9蛋白中,xCas9表现出最广泛的PAM兼容性.
- 与SpCas9相比,xCas9具有显著更高的DNA特异性和较低的目标外活性.
结论:
- 设计的xCas9扩展了CRISPR系统的DNA定位能力.
- 这种变体支持在人类细胞中的多种应用,精度更高.
- 扩展PAM兼容性不需要与Cas9编辑效率或DNA特异性进行权衡.
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