Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9.

CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.