通过CRISPR适应的综合基因组乱揭示了抗生素敏感性的复杂遗传
Wenyan Jiang1, Panos Oikonomou1, Saeed Tavazoie1
1Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Department of Systems Biology, Columbia University, New York, NY 10032, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Cell
|February 29, 2020
概括
通过CRISPR适应媒介的库制造 (CALM) 从细菌细胞中创建了全面的CRISPRi库,大大降低了成本,提高了基因功能研究和发现抗生素耐药性机制的灵敏度.
科学领域:
- 分子生物学
- 遗传学
- 微生物学
背景情况:
- 全基因组的CRISPR屏对于基因功能研究来说很强大,但却受到昂贵的低多样性指导RNA库的限制.
- 目前合成指导RNA的方法昂贵,耗时,劳动密集.
研究的目的:
- 开发一种新的,具有成本效益的方法来产生高度多样化的CRISPR干扰库 (CRISPRi).
- 提高CRISPR查的敏感性和范围,以识别新的遗传决定因素,如抗生素耐药性.
主要方法:
- 利用Streptococcus pyogenes的CRISPR-Cas适应机制开发了CRISPR适应介导的库制造 (CALM).
- CALM使用细菌细胞作为工厂,生产成千上万个不同的crRNA,针对95%的基因组部位.
- 代CRISPR适应被用来生成双crRNA库来研究双基因扰乱.
主要成果:
- 产生了高度全面的CRISPRi库,平均每基因有超过100个不同的crRNA,使得细微的转录抑制分析成为可能.
- 通过增强的CRISPRi查成功发现了新型抗生素耐药性决定因素.
- 使用代CALM创建了超过10万个双基因扰动,揭示了抗生素耐药性进化的历史偶然性.
- CALM 绕过了传统的 gRNA 合成和克隆,使野生型细菌能够生成库.
结论:
- CALM提供了一个可扩展,具有成本效益和高效的方法来生成多样化的CRISPRi库.
- 这种方法显著提高了CRISPR查的灵敏度和适用性,特别是在具有挑战性的细菌系统中.
- CALM有助于研究复杂的遗传相互作用和进化过程,例如逐步获得抗生素耐药性.
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