适应CRISPR相关的转基因用于快速和高通量逆遗传学.
David W Basta1,2,3, Franz G Zingl2,3, Yiyan Yang4
1Department of Pathology, Brigham and Women's Hospital, Boston, MA, 02115, USA.
bioRxiv : the preprint server for biology
|November 24, 2025
概括
通过使用CRISPR关联转体子 (CAST) 和指导RNA条形码,MultiCAST可实现高通量细菌遗传查. 这个平台简化了基因组工程,并有效地识别了特定条件的健身基因.
科学领域:
- 分子生物学分子生物学
- 基因组学就是基因组学.
- 合成生物学 合成生物学
背景情况:
- 与CRISPR相关的转子子 (CAST) 是细菌基因组工程的可编程工具.
- 现有的CAST系统没有针对高通量遗传查应用进行优化.
研究的目的:
- 开发MultiCAST,一个简化的平台,用于快速,可扩展,指导RNA导向的转子子插入细菌.
- 为了实现聚合,高通量基因选,使用片序列测序进行分子条形编码.
- 确定影响CAST转化效率的因素,并优化选协议.
主要方法:
- 结合性输送的等离子体编码CAST机械和一个迷你转子与指导RNA.
- 使用指导RNA序列作为分子条形码进行聚合查.
- 开发一种机器学习模型,根据目标序列特征来预测活性导向RNA.
- 研究核相关蛋白H-NS在CAST活动中的作用.
主要成果:
- 多重CAST可实现单步,针对性转子子插入,用于可扩展的遗传选.
- 优化的结合比可以最大限度地减少导向-转位子交叉声.
- 机器学习模型准确地预测了活性导向RNA.
- H-NS被确定为CAST活动的抑制剂,解释了可变插入频率.
- 在大肠杆菌中进行的大规模查,确定了特定条件的适应性基因.
结论:
- MultiCAST显著提高了细菌基因组规模功能屏幕的可访问性,速度和吞吐量.
- 该平台有助于识别具有特定条件健身效应的基因.
- MultiCAST可以适应各种细菌物种,扩大其在研究和生物技术中的应用.
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