UNIVERSITY PARK, Pa. — A technology patented by a Penn State researcher to enhance the capabilities of the genome-editing tool known as CRISPR/Cas will have potential commercial applications as the result of a recently signed licensing agreement.
Inscripta Inc., a global life science technology company, executed an agreement with Penn State’s licensing entity, the Penn State Research Foundation, to license the technology developed by Yinong Yang, professor of plant pathology in the College of Agricultural Sciences, for use with Inscripta's trademarked MAD7 nuclease. The company developed the MAD7 nuclease and began providing free access to the enzyme in December 2017 for certain research and development activities.
CRISPR has become an increasingly critical gene-editing tool, with broad applications in agriculture, medicine, industrial biotechnology and basic biological research. Using guide RNA (gRNA) to direct a nuclease — or DNA-cutting enzyme — such as Cas9 or MAD7 to a targeted region of DNA, the technology can efficiently and precisely modify an organism's genome. The resulting modifications can include a deletion, insertion, substitution or replacement of specific DNA pieces, thereby promoting or disabling gene function and associated traits.
According to Yang, one of the major advantages of CRISPR technology is its flexibility for multiplex gene editing. He explained that many potential applications of genome editing require the manipulation of more than one gene or target site, creating a need to deploy multiple gRNAs simultaneously. Previous methods of accomplishing this can be cumbersome or inefficient, typically requiring the stacking of multiple gRNA expression "cassettes" together.
"The need for simultaneous expression of multiple gRNAs was a bottleneck that prevented us from realizing the full potential of CRISPR technology," he said.
Working initially with rice as a model system, Yang's laboratory overcame this limitation by hijacking the plant cells' native processing system for transfer RNA, or tRNA. Among the normal functions of tRNA is delivering amino acids for protein synthesis within cells.
"We designed a synthetic gene with eight tandem repeats of tRNA and gRNA linked together," Yang said. "We found that tRNA-processing enzymes precisely cleaved this architecture at the tRNA-gRNA junction, efficiently releasing multiple gRNAs that can direct the CRISPR enzyme to multiple sites for genome editing."
Yang's research team used this single expression cassette to achieve the desired genetic modifications with a success rate of up to 100%, which is significantly more efficient than other existing methods, Yang noted. His group also has applied the method successfully on human cell cultures, fungi and other plant species, including tomato, potato and Arabidopsis.
"Because the tRNA-processing system exists in virtually all organisms, this strategy has been used broadly by many researchers to enhance CRISPR-based multiplex genome editing in plants, animals, humans and microbes for agricultural, medical and industrial biotech applications," Yang said.
He added that U.S. and foreign patents have been awarded for his lab's technology, which Penn State also previously licensed to a major agribiotech company for its application in crop improvement.
Inscripta intends to explore the use of Penn State’s tRNA technology in its research and development activities to improve the efficiency of gene editing for certain applications using Inscripta’s digital genome engineering platform Onyx, which is a registered trademark.