A simplified strategy for titrating gene expression reveals new relationships between genotype, environment, and bacterial growth

Author:

Mathis Andrew D1,Otto Ryan M1,Reynolds Kimberly A12ORCID

Affiliation:

1. The Green Center for Systems Biology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

2. Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

Abstract

Abstract A lack of high-throughput techniques for making titrated, gene-specific changes in expression limits our understanding of the relationship between gene expression and cell phenotype. Here, we present a generalizable approach for quantifying growth rate as a function of titrated changes in gene expression level. The approach works by performing CRISPRi with a series of mutated single guide RNAs (sgRNAs) that modulate gene expression. To evaluate sgRNA mutation strategies, we constructed a library of 5927 sgRNAs targeting 88 genes in Escherichia coli MG1655 and measured the effects on growth rate. We found that a compounding mutational strategy, through which mutations are incrementally added to the sgRNA, presented a straightforward way to generate a monotonic and gradated relationship between mutation number and growth rate effect. We also implemented molecular barcoding to detect and correct for mutations that ‘escape’ the CRISPRi targeting machinery; this strategy unmasked deleterious growth rate effects obscured by the standard approach of ignoring escapers. Finally, we performed controlled environmental variations and observed that many gene-by-environment interactions go completely undetected at the limit of maximum knockdown, but instead manifest at intermediate expression perturbation strengths. Overall, our work provides an experimental platform for quantifying the phenotypic response to gene expression variation.

Funder

Gordon and Betty Moore Foundation

National Institutes of Health

Publisher

Oxford University Press (OUP)

Subject

Genetics

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