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

• Published in: Nature biotechnology Vol. 37; no. 11; pp. 1294 - 1301
• Main Authors: Reis, Alexander C., Halper, Sean M., Vezeau, Grace E., Cetnar, Daniel P., Hossain, Ayaan, Clauer, Phillip R., Salis, Howard M.
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.11.2019; Nature Publishing Group; Springer Nature
• Subjects: 60 APPLIED LIFE SCIENCES; 631/553/552; 631/61/318; 631/61/338/552; Acid production; Agriculture; Amino acids; Amino Acids - biosynthesis; Analysis; Arrays; Bacterial genetics; Bioinformatics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biosynthesis; Biotechnology; Cellular stress response; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR/Cas9; Datasets; Deoxyribonucleic acid; Design; Discriminant analysis; DNA; DNA biosynthesis; E coli; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Editing - methods; Gene expression; Gene Expression Regulation, Bacterial; Gene regulation; Gene sequencing; Gene silencing; Genes; Genomic instability; Genomics; Learning algorithms; Letter; Life Sciences; Machine Learning; Metabolic Engineering; Mutation; Nucleotide sequence; Phenotypes; RNA sequencing; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Succinic acid; Succinic Acid - metabolism; Synthetic Biology; United States
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/s41587-019-0286-9

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. Stable multi-gene CRISPR interference using nonrepetitive genetic parts and system design.