为了更高阶的组合色素扰动而设计的CRISPR-Cas12a.
C C-S Hsiung1,2,3,4, C M Wilson2,3,4,5, N A Sambold4
1Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
Nature biotechnology
|May 17, 2024
概括
研究人员开发了一种新的CRISPR干扰 (CRISPRi) 工具,multiAsCas12a-KRAB,可以同时针对多个基因组位点. 这一进步促进了对遗传相互作用和调节元件功能的高效聚合查.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 多重复合的遗传干扰对于研究基因相互作用至关重要.
- 克里斯普尔干扰 (CRISPRi) 是一种无基因毒性的方法,用于在聚合屏幕中扰乱遗传元素,特别是非编码的基因元素.
- 目前的CRISPRi方法仅限于针对每个细胞的几个基因组位点.
研究的目的:
- 设计一种能够进行多重复制的转录抑制的新型CRISPR干扰系统.
- 提高使用CRISPRi的组合基因查的效率和范围.
- 通过组合性扰动,使调节元件的发现和功能剖析成为可能.
主要方法:
- 一种具有稳定突变 (R1226A) 的Acidaminococcus Cas12a (AsCas12a) 变种 (multiAsCas12a) 的工程,用于增强DNA切割.
- 多AsCas12a与KRAB效应器域的融合,用于强大的转录抑制.
- 开发6个复合CRISPRRNA (crRNA) 数组,用于多重化CRISPRi.
- 多AsCas12a-KRAB系统在人体细胞中高通量聚合屏幕中的应用.
主要成果:
- 与DNase-dead AsCas12a-KRAB融合相比,多AsCas12a-KRAB系统显示了较好的CRISPRi活动.
- 该工程系统成功地挽救了以前不活跃的lentivirally传递crRNAs的活动.
- multiAsCas12a-KRAB支持多重复合的CRISPRi,使用聚合屏幕中的6个复合crRNA数组.
- 该系统被用来识别增强元件,并分析 cis 调节元件的组合函数.
结论:
- 多AsCas12a-KRAB系统为多重转录干扰提供了一个强大的工具.
- 这项技术使许多染色质扰乱组合的高效小组测试能够用于生物发现.
- 开发的框架通过允许大规模的组合性扰动分析,推进了基因查和工程领域.
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