When good guides go bad: empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread...

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease in which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption that inactive gRNAs are rare persists in the E. coli community. To resolve these contradictions, we evaluated the activity of nearly 500,000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional.These nonfunctional gRNAs exhibit strong spacer self-interaction, which can be either excluded using a simple design rule or "repaired" during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million E. coli gRNAs that have been empirically evaluated in vivo as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally.