Long-Term Rewritable Report and Recording of Environmental Stimuli in Engineered Bacterial Populations

DNA writing (living sensing recorders) based whole-cell biosensors can capture transient signals and then convert them into readable genomic DNA changes. The primitive signals can be easily obtained by sequencing technology or analysis of protein activity (such as fluorescent protein). However, the functions of the current living sensing recorders still need to be expanded, and the difficulty of rewriting in complex biological environments has further limited their applications. In this study, we designed a long-term rewritable recording system using a CRISPR base editor-based synthetic genetic circuit, named CRISPR-istop. This system can convert stimuli into changes in the fluorescence intensity (reporter) and single-base mutations in genomic DNA (recording). Furthermore, we updated the biological circuit through the strategy of coupling the single-base mutation (record site) and the loss-of-function of the targeted protein (translation stopped by stop codon introduction), and we can remove edited bacteria from a population through selective sweeps upon applying a selective pressure. It successfully conducted the rewritable reporter and recording of the nutrient arabinose and pollutant arsenite with two rounds of continuous operation (10 passages/round, 12 h/passage). These observations indicated that the CRISPR-istop system can report and record stimuli over time; moreover, the recording can be manually erased and rewritten as needed. This method has great potential to be extended to more complicated recording systems to execute sophisticated tasks in inaccessible environments for synthetic biology and biomedical applications, such as monitoring disease-relevant physiological markers or other molecules.