CRISPR-Cas9 Bioremediation

A new era for environmental recovery

Population growth and industrialisation has led to harmful pollution of our air, freshwater, oceans, and soil. Water quality has worsened as a result of human activity due to mining, fracking and the processing of metals from the likes of steel mills, battery companies and electricity generation. Industrial effluents like petroleum products, polythenes, and heavy metals damage the environment and are a major cause for concern. (1)

Although most of these compounds exist in nature, they often decompose slowly or not at all and can have a severe impact on wildlife, biodiversity, ground water and human health. (1) CRISPR-Cas9 can help provide a solution to address this problem by allowing desired traits to be introduced into and enhanced in microorganisms for bioremediation. (2)

Bioremediation is a cost-effective and practical solution that uses microorganisms and microbial enzymes to detoxify contaminants in soil, water and other environments. (3)

Microorganisms are a vital component of plant growth, insect control, soil conservation, nutrient recycling, and pollutant reduction. In order to partake in these key functions microorganisms have evolved to enzymatically break down or adsorb organic and inorganic compounds to produce sources of carbon for energy, synthesise useful metabolites and or lower the toxicity of their environment. (4) By taking advantage of this adaptation, a wide variety of microorganisms can deployed for bioremediation. See table below:

Some microbes can grow at temperatures as low as −196oF/-126oC and as high as 1200oF/650oC. This adaptability and versatility makes them ideal candidates for remediation [6]. Many genetic traits contribute to the ability of these microbes to function at these temperatures, but other factors are also critical, including bacterial growth rate, high ATP usage, presence of oxygen, soil moisture content, nutrient supply, pH, and competitive species such as protozoa and bacteriophages. All of these factors must be optimized in order to effect the most efficient bioremediation. Enter CRISPR-Cas9.

CRISPR-cas9 is a remarkable genome editing tool that has been revolutionizing research in virtually every industry and bioremediation is no exception. (2) Through genome editing, a gene can either be introduced, deleted, or up- and down-regulated at a specific site within an organism (7).

CRISPR-Cas9 is an incredibly efficient tool to introduce, “swap”  or remove genes – allowing for aggregation of desirable characteristics into a single organism, including specific genes required to break down, adsorb and/or metabolise environmental pollutants. This process alters the native genome in a very precise manner to change the physiological characteristics of an individual microbe.

A report by Martínez-García et al. on the systematic deletion of 11 non-adjacent genomic regions in a Pseudomonas strain is a unique example of genome streamlining in a popular bacterial host with well-defined biodegradation capabilities (5). 300 genes were eliminated from the strain’s genome using homologous recombination after in vivo DNA cleavage.  This is an excellent demonstration of how editing a microbe’s genome can enhance a multitude of desirable traits and remove genes that could inhibit the efficiency for remediation. Moreover, due to the higher NADPH/NADP+ ratio, the strains also better tolerated endogenous oxidative stress, a property that provides a crucial advantage for catalysing harsh biodegradation reactions such as aerobic dehalogenation of chlorinated pollutants (5).

“Super” bioremediation solutions can be designed using CRISPR-Cas9 to combine desirable genetic traits from one species, such as Cunninghamella elegans’ ability to chelate lead ions, with Thermus thermophilus’ ability as a carrier to withstand extreme temperatures, creating excellent purpose-specific candidates. (11,15)

CRISPR-Cas9’s versatility allows for the alteration or insertion of virtually any known gene in any organism. This toolkit enables the creation of optimised bio-remediators that have the perfect characteristics to reverse potentially disastrous pollution events such as chemical and oil leaks, safeguarding industrial manufacturing, the water table and the environment.

1. Gaur, V.K. et al. (2020) “Assessing the impact of industrial waste on environment and mitigation strategies: A comprehensive review,” Journal of Hazardous Materials, 398, p. 123019. Available at: https://doi.org/10.1016/j.jhazmat.2020.123019.
2. Bhattacharjee, G., Gohil, N. and Singh, V. (2020) “Synthetic Biology Approaches for Bioremediation,” Bioremediation of Pollutants, pp. 303–312. Available at: https://doi.org/10.1016/b978-0-12-819025-8.00014-4.
3. Tripathi, M.; Singh, D.N.; Prasad, N.; Gaur, R. Advanced Bioremediation Strategies for Mitigation of Chromium and Organics Pollution in Tannery. In Rhizobiont in Bioremediation of Hazardous Waste; Kumar, V., Prasad, R., Kumar, M., Eds.; Springer: Singapore, 2021; pp. 195–215.
4. Tripathi, M.; Gaur, R. Bioactivity of soil microorganisms for agriculture development. In Microbes in Land Use Change Management; Singh, J.S., Tiwari, S., Singh, C., Singh, A.K., Eds.; Elsevier: Amsterdam, The Netherlands; Academic Press: Cambridge, MA, USA, 2021; pp. 197–220.
5. Martínez-García, E. et al. (2014) “Pseudomonas 2.0: Genetic upgrading of P. Putida KT2440 as an enhanced host for heterologous gene expression,” Microbial Cell Factories, 13(1). Available at: https://doi.org/10.1186/s12934-014-0159-3.
6. Hussain, A.; Rehman, F.; Rafeeq, H.; Waqas, M.; Asghar, A.; Afsheen, N.; Rahdar, A.; Bilal, M.; Iqbal, H.M. In-situ, Ex-situ, and nano-remediation strategies to treat polluted soil, water, and air—A review. Chemosphere 2022, 289, 133252.
7. Jinek, M. et al. (2012) “A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity,” Science, 337(6096), pp. 816–821. Available at: https://doi.org/10.1126/science.1225829.
8. KrabHüsken,L.ProductionofCatechols:MicrobiologyandTechnology.Ph.D.Thesis,WageningenUniversityandResearch, Wageningen, The Netherlands, 2002.
9. Miri, S.; Rasooli, A.; Brar, S.K.; Rouissi, T.; Martel, R. Biodegradation of p-xylene—A comparison of three psychrophilic Pseudomonas strains through the lens of gene expression. Environ. Sci. Pollut. Res. 2022, 29, 21465–21479.
10. Kunze, M.; Zerlin, K.F.; Retzlaff, A.; Pohl, J.O.; Schmidt, E.; Janssen, D.B.; Reineke, W. Degradation of chloroaromatics by Pseudomonas putida GJ31: Assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome. Microbiology 2009, 155, 4069–4083.
11. Duc, H.D.; Hung, N.V.; Oanh, N.T. Anaerobic Degradation of Endosulfans by a Mixed Culture of Pseudomonassp. and Staphylococcus sp. Appl. Biochem. Microbiol. 2021, 57, 327–334.
12. Gaur, V.K.; Tripathi, V.; Manickam, N. Bacterial-and fungal-mediated biodegradation of petroleum hydrocarbons in soil. In Development in Wastewater Treatment Research and Processes; Elsevier: Amsterdam, The Netherlands, 2022; pp. 407–427.
13. KharangateLad,A.;D’Souza,N.C.CurrentApproachesinBioremediationofToxicContaminantsbyApplicationofMicrobial Cells; Biosurfactants and Bioemulsifiers of Microbial Origin. In Rhizobiont in Bioremediation of Hazardous Waste; Springer: Singapore, 2021; pp. 217–263.
14. Raquel, S.; Natalia, G.; Luis Fernando, B.; Maria Carmen, M. Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by a wood-degrading consortium at low temperatures. FEMS Microbiol. Ecol. 2013, 83, 438–449.
15. Adalsteinsson, B.T. et al. (2021) “Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable cas9 variant,” Scientific Reports, 11(1). Available at: https://doi.org/10.1038/s41598-021-89029-2.