Chromatin-Optimized Crispr Activation for Enhanced Pollutant Degradation in Rhodococcus Consortia
DOI:
https://doi.org/10.65150/EP-jnsrr/V2E8/2026-01Keywords:
Bioremediation, Crispra, Chromatin, Microbial Consortia, Pollutant Degradation, Synthetic MetabolismAbstract
The system we are introducing allows for dynamic transcriptional targeting through an optimized CRISPR activation (CRISPRa) system that will improve the ability of Rhodococcus consortia to degrade pollutants. The majority of metabolic engineering approaches to date do not take into consideration chromatin silencing as a source of surprise that may result in unexpected transcriptional regulation of metabolic pathways within non-model organisms. The proposed approach combines chromatin accessibility profiling with a conditional variational autoencoder (cVAE) model to identify Nuclease-depleted regions (NDR's) that are chronically available for interaction with dCas9-VPR complexes to activate target downstream GbN for sustained transcriptional activation of the GbN in response to dynamic increases in pollutant concentration. Where stable NDR's proximal to key metabolic enzyme genes are identified by the cVAE as being stable chromatin, an NDR's proximity to a key metabolic enzyme locus can be used to identity likely site(s) for placement of dCas9-VPR complexes for sustained activation of the enzyme system. To prioritize the metabolically active NDR's, a provided Promoter Accessibility Score was calculated to account both local and distal regulatory controls on each NDR to identify the optimum transcriptional initiating site (TIS) for a final targeted dCas9-VPR complex placement. A plasmid-cloned, delivered CRISPRa system was developed to validate pathway function; this system provided dCas9-VPRtmp and gRNA's that were formulated for pollution-specific use, coupled with Codon-Optimized effectors, for use in metabolic pathway activation and evaluation by quantitative measures, including improvement in degradation rates of hydrocarbons as well as poly-aromatic hydrocarbons through a rated time course experiment. Ultimately, the proposed cVAE based CRISPRa system will be able to provide real-time adaptability to changes in the environment, setting the cVAE based CRISPRa apart from static dual-promoter based systems to create a greater degree of Flexibility in the metabolic response of Microbial consortia associated with biodegradation as well as bioremediation applications. This work demonstrates the relationship between Chromatin Biology and Synthetic Metabolism and provides a scalable, rapidly adaptable bioremediation system that can be ascribed with a robust response capability to fluctuations in Environmental input and ongoing target gene product variation, such as are present within non-model organisms over extended time periods, a feature that is critical to achieving desirable levels of genetic stability during bioprocessing for bioremediation and biodegradation.
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Copyright (c) 2026 Sebastian, Jeremia D., Akpadolu, Chidinma B, Benin, Sandra, Okoye, Rosemary, Owusu, Nana H. (Author)
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