Empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread functionality and rules for gRNA design

Journal Article · Mon Jun 01 00:00:00 UTC 2026 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkag548 · OSTI ID:3374772

Kammerdiener, Elise  ^[1]; Harrison, Riley ^[2]; Charles, S'Khaja ^[3]; Klingeman, Dawn Marie  ^[1]; Wiser, Timothy ^[4]; Eckert, Carrie  ^[1]; Alexander, William  ^[1]

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1. ORNL
2. Brown University, Providence, RI
3. University of Chicago
4. Truman State University

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with 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 persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 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 either be 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 empirically evaluated E. coli gRNAs 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. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE)
• Grant/Contract Number: AC05-00OR22725
• OSTI ID: 3374772
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 11 Vol. 54
• Country of Publication: United States
• Language: English