Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.
Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA.
Nat Biotechnol. 2019 Nov;37(11):1294-1301. doi: 10.1038/s41587-019-0286-9. Epub 2019 Oct 7.
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.
工程细胞表型通常需要调节许多基因。当使用 CRISPR 干扰时，由于存在重复 DNA 序列，共表达许多单引导 RNA(sgRNA)会引发遗传不稳定性和表型丧失。我们通过稳定共表达非重复超长 sgRNA 阵列(ELSAs)内的 22 个 sgRNA，同时通过高达 3500 倍的抑制多达 13 个基因。我们应用生物物理建模、生化特性分析和机器学习来开发非重复遗传元件工具箱，包括 28 个与 Cas9 结合的 sgRNA 手柄。我们根据算法规则设计 ELSAs，该规则量化了 DNA 合成复杂性、sgRNA 表达、sgRNA 靶向和遗传稳定性，然后根据算法规则将非重复遗传元件组合在一起。使用 ELSAs，我们在大肠杆菌中创建了三种高度选择性的表型，包括将代谢重定向以将琥珀酸产量提高 150 倍、敲低氨基酸生物合成以创建多营养缺陷型菌株以及抑制应激反应以将持久性细胞形成减少 21 倍。ELSAs 可用于代谢工程和合成生物学应用中的许多基因的同时稳定调控。
