几乎没有PAM的CRISPR-Cas9基因组向
Russell T Walton1,2, Kathleen A Christie1,2,3, Madelynn N Whittaker1,2
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
概括
研究人员设计了CRISPR-Cas9变体,SpG和SpRY,以克服原空间器相邻动机 (PAM) 的限制. 这些先进的基因组编辑工具扩大了精确基因研究和疾病变异生成的准能力.
科学领域:
- 分子生物学
- 遗传学
- 生物技术
背景情况:
- CRISPR-Cas9基因编辑依赖于原空间器相邻动机 (PAM) 的识别.
- 这一PAM要求限制了可用于基因组操纵的目标DNA序列.
- 现有的CRISPR-Cas9系统,如*Streptococcus pyogenes* Cas9 (SpCas9),受到其特定的PAM序列 (例如NGG) 的限制.
研究的目的:
- 设计 SpCas9 变种以消除或减少对正规 NGG PAM 的依赖.
- 为更广泛的基因组编辑应用扩大CRISPR-Cas9的准范围.
- 开发新的工具来产生以前无法获得的遗传变异,包括与疾病相关的变异.
主要方法:
- 用SpCas9的蛋白质工程来创建具有改变PAM特异性的变体.
- 开发针对NGN PAM的SpG变种.
- 进一步优化以创建SpRY变种,针对NRN和NYN PAM.
- 用于评估 SpG 和 SpRY 在人体细胞中的核酶和基编辑器活动.
主要成果:
- 工程 SpG 变种成功针对 NGN PAM 序列.
- 开发的SpRY变种表现出广泛的PAM向,包括NRN和NYN动机.
- 在人类细胞中,SpRY在NRN PAM位点表现出强烈的活性,在NYN PAM位点表现出实质性的活性.
- SpG 和 SpRY 能够产生与疾病相关的基因变异,而野生型 SpCas9 无法实现.
结论:
- SpG和SpRY显著扩大了CRISPR-Cas9技术的目标范围.
- 这些工程变异克服了PAM的局限性,使得高分辨率的基因组编辑成为可能.
- 开发的工具对于包括遗传疾病研究在内的各种应用非常有价值.
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