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Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes by Jian Wang; Chenyi Li; Tian Jiang; Yajun Yan is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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With rapid advances in the development of metabolic pathways and synthetic biology toolkits, a persisting challenge in microbial bioproduction is how to optimally rewire metabolic fluxes and accelerate the concomitant high-throughput phenotype screening. Here we developed a biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening approach that combines a titratable mismatch CRISPR interference and a biosensor mediated screening for high-production phenotypes in Escherichia coli. We first developed a programmable mismatch CRISPRi that could afford multiple levels of interference efficacy with a one-pot sgRNA pool (a total of 16 variants for each target gene) harboring two consecutive random mismatches in the seed region of sgRNA spacers. The mismatch CRISPRi was demonstrated to enable almost a full range of gene knockdown when targeting different positions on genes. As a proof-of-principle demonstration of the BATCH screening system, we designed doubly mismatched sgRNA pools targeting 20 relevant genes in E. coli and optimized a PadR-based p-coumaric acid biosensor with broad dynamic range for the eGFP fluorescence guided high-production screening. Using sgRNA variant

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Author

Jian Wang; Chenyi Li; Tian Jiang; Yajun Yan

Publisher

Elsevier BV

Published

2023

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)