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通过工程CRISPR-Cas12a对功能基因组学进行更高阶组合色素扰动

C C Hsiung, C M Wilson, N A Sambold

bioRxiv : the preprint server for biology

|October 2, 2023

概括

研究人员开发了multiAsCas12a,这是一种新的CRISPR干扰工具,可以实现更高阶的组合基因向. 这一创新推动了功能性基因组学,允许生物发现更复杂的扰动.

科学领域:

遗传学遗传学是一种遗传学.
分子生物学分子生物学
生物技术是生物技术.

背景情况:

多重复合的遗传乱对于理解基因相互作用至关重要.
克里斯普尔干扰 (CRISPRi) 提供了一种非遗传毒性方法来扰乱遗传元素,特别是非编码区域.
目前的CRISPRi聚合查仅限于扰乱每个细胞的1-3个基因组位点.

研究的目的:

设计一种新的CRISPR干扰工具,用于更高阶 (大于3) 的基因组位点的组合定位.
使用CRISPRi技术增强功能基因组学屏幕的能力.

主要方法:

设计了一种 *Acidaminococcus* Cas12a 变种,称为多重转录干扰 AsCas12a (multiAsCas12a),具有特定的 R1226A 突变.
测试了multiAsCas12a用于CRISPR干扰活性,使用聚合屏幕中的CRISPRRNAs (crRNAs) 的高顺序多重组数组.
在组合CRISPRi向中对现有Cas12a变异的评估性能.

主要成果:

在组合CRISPRi向中,multiAsCas12a在现有Cas12a变体上显著提高了性能.
在高通量聚合屏幕中成功使用了6个复数crRNA数组库.
能够发现新的增强元件和剖析 cis-调节元件组合函数.

结论:

设计的多AsCas12a变体使高效的,更高阶的组合CRISPRi选成为可能.
该工具为生物发现和工程提供了一个强大的组测试框架.
方便对众多染色质扰乱组合进行有效的测量.

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