By Eric Rhodes, CEO at ERS Genomics
CRISPR/Cas9 genome editing is fast becoming the essential tool for industrial microbial production and chemical synthesis by virtue of its speed, precision, and versatility. CRISPR can drastically improve the efficiency of industrial biotechnology through enzyme engineering and pathway modification and is already being put to work to change chemical manufacturing. Here we run through some of the most exciting applications of CRISPR.
Challenges in industrial biotechnology
Microorganisms have long been used in the synthesis of products that are useful for human societies. Yeast, for example, have been used in the production of beer and wine for millennia, with the underlying process of fermentation uncovered in the 18th century by Louis Pasteur, who was the first to show how yeast convert sugar into alcohol.1 Following the discovery of penicillin, industrial microbiology took off as the antibiotic was mass-produced to support the war effort.2
Today, microbes are being used to synthesize diverse products, from foods and drugs to cosmetics and biofuels. By 2026, the global synthetic biology market, covering tools, technologies, and applications, is expected to grow to more than $30 billion, with medical and industrial bioproduction leading the way.
Yet, challenges remain in translating advances in bioengineering into efficient mass production of products. Engineering new microbial strains for production is often time-consuming and expensive, requiring extensive optimization. This is further hindered by the speed at which microorganisms grow and the time it takes for complex biosynthetic reactions to complete, which can slow production and limit yields.
Other complicating factors include navigating the complexity of cellular metabolism, the requirement for large amounts of substrate, which may include precious natural resources, and excess byproduct production.3
CRISPR: a game-changing tool
The entry of CRISPR/Cas9 gene editing technology onto the commercial market has made it an indispensable tool in industrial microbiology. Discovered in bacteria by Nobel Prize winners Emmanuelle Charpentier and Jennifer Doudna, CRISPR/Cas9 makes precise cuts in DNA, using natural cellular repair mechanisms to inactivate a gene, delete a section of DNA, or even insert new genetic information.
On average, a bacterial genome contains around 30 biosynthetic gene clusters, potentially encoding 30 different natural products.4 CRISPR/Cas9 gene engineering is expanding the range of these products that are accessible to humans, by increasing the number of microbial strains that can be effectively used in industrial production and making more of their metabolic pathways suitably efficient for large-scale production.5 The possibilities of this technology for industrial biotechnology are almost limitless, as it combines precision with the versatility to manipulate complicated cellular processes in cells in a range of different ways.
CRISPR also enables multiplexing, so that multiple genes can be modified at the same time. It is easy to use, thanks to accessible licensing and a surge of third-party companies offering “ready to go” CRISPR-based genome engineering platforms.
Top CRISPR/Cas9-modified biosynthetic pathways
CRISPR/Cas9 gene editing is a powerful way of improving biosynthesis for organic and inorganic chemicals, and novel products that are not naturally produced by the host cell. Some of the top compounds being made using CRISPR-modified cells include carotenoids, citric acid, 1,3-propanediol, phenylethanol, and squalene.
Carotenoids are yellow, orange, and red pigments found naturally in a range of foods and plants. They are important nutrients and are also used to produce food colorings and fragrances for a variety of products. CRISPR/Cas mutation libraries have been used to identify more efficient enzyme variants in the production of carotenoids, resulting in an 11-fold improvement in carotenoid production in yeast.6
Citric acid is naturally found in citrus fruits but billions of tons are manufactured each year for use as an acidifier and flavoring agent. CRISPR/Cas9 has been used to increase the efficiency of its production in its workhorse organism, Aspergillus niger.7
CRISPR has also enhanced the production of 1,3-propanediol, an important building block in polymer production, used in a range of industrial products and as a solvent. Using CRISPR, the production of this compound has been increased by almost 50% in the bacterium Klebsiella pneumoniae.8
Phenylethanol is a fragrant alcohol used in food and cosmetics. CRISPR has been used to engineer the overexpression of this floral-scented compound by a stress-tolerant strain of yeast. The same strategy could be used to produce other aromatic compounds in this host.7
Squalene, a compound found in high quantities in shark liver oil, is an ingredient in cosmetics and used as an adjuvant in vaccines. CRISPR has been used to modify bacteria to produce squalene from glucose at high efficiencies, supporting more sustainable production.9
Enzyme engineering using CRISPR
One of the major applications of CRISPR in industrial biotechnology is the engineering of enzymes. CRISPR-guided DNA polymerases can be used to target nucleotides for mutagenesis, offering an extremely high targeted mutation rate and introducing a range of novel mutations that may be beneficial to enzyme function.
This approach has been used in E. coli, where the engineering of a new enzyme involved in amino acid synthesis increased the production of tryptophan—widely used in the food, feed, and medical industries by almost 40%.4,14
CRISPR can also be used to optimize metabolic pathways by deleting, knocking down, or overexpressing genes. In a groundbreaking example, CRISPR/Cas9 was used to delete eight genes in a fatty acid production pathway in yeast, resulting in a 30-fold increase in free fatty acid production in a matter of days.15
Other approaches include knocking down a competing pathway using transcriptional repression (CRISPRi), which has been used to enhance the production of various compounds. The advantage of this approach is that the strength of repression can be modified depending on the combinations of genes targeted.
Similarly, transcriptional activation (CRISPRa) can be used to upregulate genes to increase production. Both CRISPRi and CRISPRa methods are highly useful for multiplex engineering, whereby several pathways in cellular metabolism can be finetuned at the same time.
The versatility of the technology is unmatched and many companies are starting to take advantage of this. For example, U.S.-based Gingko Bioworks has applied CRISPR/Cas9 to modify cells for a wide range of purposes, partnering with pharmaceutical companies, food producers, and cosmetics companies to bring the biosynthetic potential of this powerful technology to life.
CRISPR supports more sustainable chemical production, which is a major concern for the industry at present. Through sophisticated pathway modifications, CRISPR is able to change the feedstocks that microbes are able to use. This is supporting a shift towards renewable or waste substrates and those that generate a higher yield of product.
CRISPR can also be used to modify microbes to grow at lower temperatures and, as in the case of squalene similar natural products, to generate a more sustainable source of compounds that reduces pressure on threatened plant and animal species. Biotechnology will be key to meeting the ever-growing demand for large-scale, sustainable chemical manufacturing. As current processes are optimized and new compounds come to market, CRISPR/Cas9 has a vital part to play in bringing forward this bio-industrial revolution.
- Alba-Lois, L. & Segal-Kischinevzky, C. (2010) Beer & Wine Makers. Nature Education 3(9):17.
- Buchholz, K. and Collins, J. (2013) Industrial Biotechnology. Sustainable Growth and Economic Success. Edited by Wim Soetaert and Erick J. Vandamme.
- Chen, G.-Q. (2012). New challenges and opportunities for industrial biotechnology. Microbial Cell Factories, 11(1), p.111. doi:10.1186/1475-2859-11-111.
- Chen, M., Chen, L. and Zeng, A.-P. (2019). CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli. Metabolic Engineering Communications, 9, p.e00094. doi:10.1016/j.mec.2019.e00094.
- Hille, F., Richter, H., Wong, S.P., Bratovič, M., Ressel, S. and Charpentier, E. (2018). The Biology of CRISPR-Cas: Backward and Forward. Cell, 172(6), pp.1239–1259. doi:10.1016/j.cell.2017.11.032.
- Jakočiūnas, T., Pedersen, L.E., Lis, A.V., Jensen, M.K. and Keasling, J.D. (2018). CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9. Metabolic Engineering, 48, pp.288–296. doi:10.1016/j.ymben.2018.07.001.
- Li, M., Lang, X., Moran Cabrera, M., De Keyser, S., Sun, X., Da Silva, N. and Wheeldon, I. (2021). CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus. Biotechnology for Biofuels, 14(1). doi:10.1186/s13068-020-01852-3.
- Lian, J., HamediRad, M., Hu, S. and Zhao, H. (2017). Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nature Communications, 8(1). doi:10.1038/s41467-017-01695-x.
- Park, J., Yu, B.J., Choi, J. and Woo, H.M. (2019). Heterologous Production of Squalene from Glucose in Engineered Corynebacterium glutamicum Using Multiplex CRISPR Interference and High-Throughput Fermentation. Journal of Agricultural and Food Chemistry, 67(1), pp.308–319. doi:10.1021/acs.jafc.8b05818.
- Shi, S., Qi, N. and Nielsen, J. (2022). Microbial production of chemicals driven by CRISPR-Cas systems. Current Opinion in Biotechnology, 73, pp.34–42. doi:10.1016/j.copbio.2021.07.002.
- Steele, A.D., Teijaro, C.N., Yang, D. and Shen, B. (2019). Leveraging a large microbial strain collection for natural product discovery. Journal of Biological Chemistry, 294(45), pp.16567–16576. doi:10.1074/jbc.rev119.006514.
- Tong, Z., Zheng, X., Tong, Y., Shi, Y.-C. and Sun, J. (2019). Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era. Microbial Cell Factories, 18(1). doi:10.1186/s12934-019-1064-6.
- Wang, X., Zhang, L., Liang, S., Yin, Y., Wang, P., Li, Y., Chin, W.S., Xu, J. and Wen, J. (2022). Enhancing the capability of Klebsiella pneumoniae to produce 1, 3-propanediol by overexpression and regulation through CRISPR. Microbial Biotechnology, 15(7), pp.2112–2125. doi:10.1111/1751-7915.14033.
- Wen, X., Ning, L. and Jia, Z. (2017). Production of L-tryptophan by Microbial Fermentation. Progress in Applied Microbiology.
- Zhang, Y., Wang, J., Wang, Z., Zhang, Y., Shi, S., Nielsen, J. and Liu, Z. (2019). A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nature Communications, 10(1). doi:10.1038/s41467-019-09005-3.
Eric Rhodes is CEO at ERS Genomics, which provides worldwide licensing access to the essential CRISPR/Cas9 patent portfolio for commercial use.