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

Nucleic Acids Research
Andrew D MathisKimberly A Reynolds

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 knockd...Continue Reading

References

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Citations

Apr 27, 2021·Molecular Systems Biology·Jean-Benoît LalanneGene-Wei Li
May 25, 2021·STAR Protocols·Jordi van GestelCarol A Gross

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Methods Mentioned

BETA
PCR
restriction digest
gene knockdown
gene knockout

Software Mentioned

Optimize
Jupyter
Python2
CRISPRiSeq
BLASTn
USEARCH
SciPy
Protospacer

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