In situ genotyping of a pooled strain library after characterizing complex phenotypes

• Published in: Molecular systems biology 2017-10, Vol.13 (10), p.947-n/a
• Main Authors: Lawson, Michael J, Camsund, Daniel, Larsson, Jimmy, Baltekin, Özden, Fange, David, Elf, Johan
• Format: Article
• Language: English
• Subjects: Bacteria; Chemical synthesis; CRISPR; DuMPLING; E coli; EMBO22; EMBO33; Escherichia coli - classification; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Fluorescence; Fluorescence in situ hybridization; Fluorescence microscopy; Gene expression; Gene Library; Gene sequencing; Genes; Genetic diversity; Genomes; Genotype; Genotypes; Genotyping; Genotyping Techniques - methods; Interference; Libraries; live cell; Method; microfluidic; Microfluidic Analytical Techniques; Microscopy; Phenotype; Phenotyping; Plasmids; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; RNA-mediated interference; Screens; single cell; Single Molecule Imaging - methods; strain libraries; Strains (organisms)
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.20177951

In this work, we present a proof‐of‐principle experiment that extends advanced live cell microscopy to the scale of pool‐generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single‐molecule fluorescence time‐lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter. Synopsis The DuMPLING approach extends the use of advanced live‐cell microscopy to libraries of pool generated genetically diverse strains. The method is illustrated by tracking strains over six generations and precisely quantifying gene expression at the single molecule level. A library of plasmids is pool synthesized, expressing a specific RNA barcode and an associated CRISPR interference guide RNA against different chromosomal genes. Library phenotyping is demonstrated using time‐lapse single‐molecule fluorescence microscopy of bacteria growing for many generations in a microfluidic device. The strains are genotyped in situ by six rounds of sequential fluorescent in situ hybridization probing in two colors. Graphical Abstract The DuMPLING approach extends the use of advanced live‐cell microscopy to libraries of pool generated genetically diverse strains. The method is illustrated by tracking strains over six generations and precisely quantifying gene expression at the single molecule level.