Lawrence Berkeley National Laboratory is offering an exploratory kit for its Chassis-independent, Recombinase-Assisted, Genome Engineering (CRAGE) tool. CRAGE is a novel process to chromosomally integrate and express genes or pathways in a broad range of microbes. It allows for rapid and efficient transfer and stable integration of biosynthetic pathway-encoding gene clusters of up to 60 kb into the genome. The technology has already been used at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI), a DOE Office of Science User Facility located at Berkeley Lab, to domesticate 40 strains, including those expressing biosynthetic gene clusters. It is now available for licensing.

The goal of the strain engineering Design-Build-Test-Learn (DBTL) cycle is to build stable strains that can produce industrially relevant products at large scales. This requires genome integration-based pathway expression, however, traditional approaches for pathway integration are slow and inefficient. The plasmid system, which has been widely used as a strategy for pathway optimization during the first phase of engineering, is unstable and copy numbers are inconsistent, making it challenging for scale-up.

CRAGE eliminates the plasmid-based expression phase by making genome engineering as easy as – or even easier than – plasmid-based engineering. First, a “landing pad” is inserted into the genome. The landing pad can then be used as a location for delivery of multiple types of payloads, such as individual genes, whole pathways, other genome-editing tools, and reporters. This process can be automated and significantly scaled.

“Speed in microbial engineering cycles is crucial,” said Dr. Yasuo Yoshikuni, researcher and head of the DNA Synthesis Science Program at JGI. “CRAGE makes genome scale engineering much more efficient, skipping at least 50% of the costly cycle and accelerating Design-Build-Test-Learn in industrial strain development.”

CRAGE can be used in a variety of microbes for a broad range of applications, for example, in microbiome engineering to target disease or to develop antibacterial treatments for infectious disease, or in soil microbes to produce more nitrogen for plant growth.

“Adoption of this technology can be hugely beneficial in many applications,” continued Yoshikuni. “It is easy to engineer any microbe of interest, and provides speedy and accurate results.”