Wang, S.-G. | bioRxiv Science

bioRxiv2022

Base Editing for Reprogramming Cyanobacterium Synechococcus elongatus

Global climate change demands carbon-negative innovations to reduce the concentration of atmospheric carbon dioxide (CO2). Cyanobacteria can fix CO2 from the atmosphere and can be genetically reprogrammed for the production of biofuels, chemicals and food products, making an ideal microbial chassis for carbon-negative biotechnology. However, the progress seems to be slowed down due to the lagging-behind synthetic biology toolkits, especially the CRISPR-Cas-based genome-editing tools. As such, we developed a base-editing tool based on the CRISPR-Cas system and deamination for cyanobacterium Synechococcus elongatus. We achieved efficient and precise genome editing at a single-nucleotide resolution, and identified the pure population of edited cells at the first round of selection without extra segregation. By using the base-editing tool, we successfully manipulated the glycogen metabolic pathway via the introduction of premature STOP codons to inactivate the corresponding genes. We demonstrated multiplex base editing by editing two genes at once, obtaining a nearly two-fold increase in the glycogen content. We present here the first report of base editing in the phylum of cyanobacteria, and a paradigm for applying CRISPR-Cas systems in bacteria. We believe that this work will accelerate the synthetic biology of cyanobacteria and drive more innovations to alleviate global climate change.

synthetic biology↗

bioRxiv2022

Tailoring CRISPR-Cas Immunity for the Degradation of Antibiotic Resistance Genes

The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes are key barriers preventing the spread of ARGs, they are often sources of ARGs at the same time, as ARGs may be required and accumulate in the biological treatment units. An upgrading of environmental biotechnology is imperative and urgent. ARGs confer antibiotic resistance based on the DNA sequences rather than the chemistry of DNA molecules. An ARG can be considered degraded if its sequence was disrupted. Therefore, we present here that CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, can be repurposed and tailored for the degradation of ARGs. By deploying an artificial IncP machinery, the designed system, namely VADER, can be successfully delivered via bacterial conjugation. Then, we propose a new sector for ARG degradation to be implemented as a complement to the biological units in the framework of environmental processes. In this endeavor, a prototype conjugation reactor at a 10-mL-scale was devised, and 100% of the target ARG were eliminated in the transconjugated microbes receiving VADER in the reactor. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. ImportanceAntibiotic resistance has been causing severe health problems and leading to millions of deaths in recent years. Environmental processes, especially the wastewater treatment sector, are important to barrier the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as the source of antibiotic resistance at the same time, as antibiotic resistance with its main cause antibiotic resistance genes (ARGs) may be required and accumulate in the biological treatment units, leading to the dissemination of ARGs. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to environmental biotechnology for tackling the antibiotic resistance dilemma thereof, and we propose a new sector in environmental processes specialized in ARG removal with a reactor inhabiting the CRISPR-Cas system per se. Our study provides a new angle to resolve public health issues via the implementation of synthetic biology at the process level.

synthetic biology↗