(590d) Multiplexed Information Flow Via H2O2-Facilitated Electrogenetic CRISPR for Enabling “Multilingual” Communication Among Biological Networks

We have recently reported an electrogenetic CRISPR that mediates the “conversation” between electrodes and genetic networks by tapping into the soxRS oxidative stress response regulon. In order to more efficiently traverse the abiotic and biotic domains, reduce barriers accompanying diverse biological languages, and at the same time, enable direct electronic transmission, we have created an H₂O₂-actuated eCRISPR housed in surface-assembled bacteria. It is actuated by electrode synthesized hydrogen peroxide that freely diffuses into the translating bacteria where it activates the global response regulon, oxyRS. Natural redox responders, such as the transcriptional regulator OxyR, can be directly activated by accepting (or donating) electrons. This characteristic allows the transmission of electronic information from electrodes into the cells, generating a biological response by altering gene expression. In this study, we demonstrate multiplexed activation and inhibition as well as language translation, through CRISPR-based toolkit that is controlled by an electrode-generated redox signal, H₂O₂.

To be specific, we have created an electronically tunable CRISPR-Cas9 mediated transcriptional activation (CRISPRa) system by rewiring the oxyRS regulon to inducibly express guide RNAs (gRNA) for activation of GFP and a bacterial quorum sensing (QS) signal synthase, LasI. The latter strain, capable of AI-1 production, is assembled onto the electrode by codeposition of a thiolated-PEG (PEG-SH), forming a “living electrode” that can interact with other bacterial populations when given an electric signal. We also employ electronically-controlled CRISPR interference (CRISPRi) to filter out the native oxidative stress response generated by oxyRS regulon, which, in turn, creates a positive feedback loop that enhances H₂O₂-controlled genetic activation. To demonstrate multiplex signaling, we use peroxide-facilitated eCRISPR to manipulate bacterial cell-cell signaling with a “bilingual” strain that switches between speaking different bacterial “languages”, namely two orthogonal QS signals autoinducer 1 (AI-1) and 2 (AI-2). In sum, we established an eCRISPR toolkit that accepts electronic inputs and in an aerobic environment, enhances, diminishes, or alters native biological information exchange among “conversing” microbes.