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ABSTRACT Microbes can be programmed to record participation in gene transfer by coding biological-recording devices into mobile DNA. Upon DNA uptake, these devices transcribe a catalytic RNA (cat-RNA) that binds to conserved sequences within ribosomal RNA (rRNA) and perform a trans-splicing reaction that adds a barcode to the rRNA. Existing cat-RNA designs were generated to be broad-host range, providing no control over the organisms that were barcoded. To achieve control over the organisms barcoded by cat-RNA, we created a program called Ribodesigner that uses input sets of rRNA sequences to create designs with varying specificities. We show how this algorithm can be used to identify designs that enable kingdom-wide barcoding, or selective barcoding of specific taxonomic groups within a kingdom. We use Ribodesigner to create cat-RNA designs that target Pseudomonadales while avoiding Enterobacterales, and we compare the performance of one design to a cat-RNA that was previously found to be broad host range. When conjugated into a mixture ofEscherichia coliandPseudomonas putida, the new design presents increased selectivity compared to a broad host range cat-RNA. Ribodesigner is expected to aid in developing cat-RNA that store information within user-defined sets of microbes in environmental communities for gene transfer studies. GRAPHICAL ABSTRACT 

SUMMARY Engineered microbes are being programmed using synthetic DNA for applications in soil to overcome global challenges related to climate change, energy, food security, and pollution. However, we cannot yet predict gene transfer processes in soil to assess the frequency of unintentional transfer of engineered DNA to environmental microbes when applying synthetic biology technologies at scale. This challenge exists because of the complex and heterogeneous characteristics of soils, which contribute to the fitness and transport of cells and the exchange of genetic material within communities. Here, we describe knowledge gaps about gene transfer across soil microbiomes. We propose strategies to improve our understanding of gene transfer across soil communities, highlight the need to benchmark the performance of biocontainment measuresin situ, and discuss responsibly engaging community stakeholders. We highlight opportunities to address knowledge gaps, such as creating a set of soil standards for studying gene transfer across diverse soil types and measuring gene transfer host range across microbiomes using emerging technologies. By comparing gene transfer rates, host range, and persistence of engineered microbes across different soils, we posit that community-scale, environment-specific models can be built that anticipate biotechnology risks. Such studies will enable the design of safer biotechnologies that allow us to realize the benefits of synthetic biology and mitigate risks associated with the release of such technologies.