Targeted modification of promoters

Although most genome editing efforts focus on modifications to gene coding regions, this chapter emphasizes genome editing of the upstream regulatory regions. Thoughtful editing of the promoter region will ultimately lead to improved plants, modified for more precise control of the intensity and specificity of native gene expression. In this chapter, we present an overview of the promoter or upstream regulatory region of a gene, and describe how this sequence is defined and studied. We then describe how the composition and arrangements of cis-regulatory elements within the promoter and the leading intron associated with the promoter region have been studied using classical transgenic approaches to reveal what regulatory components might be suitable for genome editing approaches. Finally, we offer some suggestions for pursuit of promoter editing and gene expression modulation, which will eventually lead to modified plants with an altered regulation of native gene expression.

Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

Escherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.