Activation. Interference. Deletion. Together, they spell AID, and they are being combined so that they may better aid metabolic engineering. AID refers to three genetic manipulations that can be accomplished simultaneously by a newly developed CRISPR system.

Ordinarily, these manipulations are carried out sequentially, so that their combined effects emerge only after a laborious multistep process is completed. CRISPR-AID, however, promises to expedite metabolic engineering projects in which multiple gene targets are perturbed—even if the targets are to be perturbed in diverse ways.

CRISPR-AID is being developed by researchers based at the Carl R. Woese Institute for Genomic Biology (IGB) at the University of Illinois. Building on CRISPR, a gene-editing approach that makes use of an RNA-guided nuclease, the researchers assembled a multifunctional metabolic engineering system.

Details about the system appeared November 22 in the journal Nature Communications, in an article entitled  “Combinatorial Metabolic Engineering Using an Orthogonal Tri-Functional CRISPR System.” The article introduces an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion. The article also describes how the system performed in the yeast Saccharomyces cerevisiae.

“This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner,” the article’s authors wrote. CRISPR-AID not only increased the production of β-carotene by three-fold in a single step, the authors reported, but also achieved a 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner.

Metabolic engineering involves engineering microorganisms to produce value-added products, such as biofuels and chemicals. This is achieved by changing or deleting the expression of genes to modify the microorganism's genome. In this process, several targets in the genome are modified to achieve specific goals.

“We can easily find several metabolic engineering targets to improve the desired phenotype,” said Jiazhang Lian, Ph.D., a visiting research associate at the IGB who is the first author of the Nature Communications article. “How to combine these beneficial genetic modifications is one of the biggest challenges in metabolic engineering.”

Traditionally, researchers test these targets individually in a series of time-consuming steps. These steps limit productivity and the yield of the final product—two crucial components in the metabolic engineering process.

The researchers decided to create a method that combines these steps. By accomplishing these steps simultaneously, scientists can explore different combinations of manipulations and discover which combination is best.

“We can now work with 20 targets,” asserted Huimin Zhao, Ph.D., the senior author of the current study and Steven L. Miller Chair of Chemical and Biomolecular Engineering at IGB. “We can implement all of these (manipulations) for each target in a combinatorial manner to find out which combination actually will give us higher productivity or yield of the final product.”

“If we compare metabolic engineering to a basketball team, we cannot build a strong team by simply putting the best players together,” Lian added. “Instead, we should try to find those who can collaborate and work synergistically.”

Their new system opens up thousands—even millions—of possibilities, which presents another logistical challenge.

They plan to find the best combinations by developing a high-throughput screening method or using a robotic system such as the iBioFAB, a system located in the IGB that automatically produces synthetic biosystems.

“I believe the combination of CRISPR-AID with high-throughput screening and iBioFAB will significantly advance the metabolic engineering field in the near future,” Lian said.

Zhao hopes to test their method on other organisms, using the same engineering principles but modifying the protocol for different organisms. Eventually, they hope to extend to the genome scale, which would be a considerable leap in the field of metabolic engineering.

“If we can do that, we can make it truly modularized and also standardize the procedure,” Zhao explained. “Then we really increase the throughput and the speed of metabolic engineering.”

“It's not just an incremental improvement,” Zhao said of CRISPR-AID. “It's a new way to do metabolic engineering.”