小柯机器人

研究发现非重复ELSA广泛抑制细菌基因
2019-10-08 14:53

美国宾夕法尼亚大学Howard M. Salis研究组的最新研究,提出使用非重复的超长单向导RNA(sgRNA)阵列(ELSAs)能够同时抑制多个细菌基因表达。2019年10月7日,《自然-生物技术》在线发表了这项成果。

他们在非重复的ELSAs中稳定共表达22个sgRNA,以同时抑制多达13个基因达3500倍。他们应用了生物物理建模,生化表征和机器学习来开发非重复性遗传部分的工具箱,包括与Cas9结合的28个sgRNA手柄。他们根据量化DNA合成复杂性,sgRNA表达,sgRNA靶向和遗传稳定性的算法规则,通过组合非重复性遗传部分来设计ELSA。使用ELSA,他们在大肠杆菌中创建了三种高度选择性的表型,包括重新定向代谢以使琥珀酸的产量增加150倍,破坏氨基酸的生物合成以产生多营养缺陷型菌株,并抑制应激反应以使持续程序细胞减少21倍。ELSA能够同时稳定地调节许多基因,以用于代谢工程和合成生物学应用。

据介绍,工程细胞表型通常需要许多基因的调节。当使用CRISPR干扰时,由于存在重复的DNA序列,共表达许多sgRNA会触发遗传不稳定和表型丧失。

附:英文原文

Title: Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays

Author: Alexander C. Reis, Sean M. Halper, Grace E. Vezeau, Daniel P. Cetnar, Ayaan Hossain, Phillip R. Clauer, Howard M. Salis

Issue&Volume: 2019-10-07

Abstract: 

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

DOI: 10.1038/s41587-019-0286-9

Source: https://www.nature.com/articles/s41587-019-0286-9

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:68.164
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex


本期文章:《自然—生物技术》:Online/在线发表

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