Watching CRISPR-Cas9 at Work in Real-Time

Detecting the activity of CRISPR gene editing tools in organisms with the naked eye and an ultraviolet flashlight is now possible using technology developed at the Department of Energy’s Oak Ridge National Laboratory.

Scientists demonstrated these real-time detection tools in plants and anticipate their use in animals, bacteria and fungi with diverse applications for biotechnology, biosecurity, bioenergy and agriculture. The team described the successful development of the UV system in Horticulture Research and their proof-of-principle demonstration in ACS Synthetic Biology.

CRISPR technologies have quickly become the primary tools of bioengineering, and new versions are continually in development. Identifying whether an organism has been modified by CRISPR technology was previously a complex and time-consuming process.

“Before this, the only way to tell if genome engineering occurred was to do a forensic analysis,” said Paul Abraham, a bioanalytical chemist and head of ORNL’s Secure Ecosystem Engineering and Design Science Focus Area. “To be successful, you would need to know what the genome looked like before it was rewritten. We wanted to design a platform where we could proactively observe CRISPR activity.”

The research team developed an efficient self-detect solution that takes advantage of the way CRISPR works to trigger the technology to reveal itself. Under normal conditions, CRISPR works by connecting with a short RNA sequence, known as the guide RNA, as it leads CRISPR to a matching DNA sequence. When the target DNA is found, CRISPR modifies the DNA by acting like tiny molecular scissors to cut through one or both strands of DNA, depending on the type of CRISPR technology in use.

Abraham likens their method to an alarm system with two components: a biosensor guide RNA that redirects CRISPR activity and a reporter protein that flags the activity. Researchers encode the two components into an organism’s DNA to enable the monitoring system.

With the self-detect system in place, the biosensor guide RNA intercepts CRISPR, preventing CRISPR from connecting with its original gene target and redirecting CRISPR to a specific DNA sequence that encodes for a nonfunctioning green fluorescent protein, or GFP. When CRISPR edits the sequence, it flips a switch that produces functioning GFP, which creates a green glow signaling CRISPR’s presence.

Because a microscope is required to see the glow from GFP, the researchers improved on their original method by replacing GFP with a similar reporter protein, called eYGFPuv, that is visible under the type of ultraviolet light commonly known as a black light.

Reference: Yuan G, Hassan MdM, Yao T, et al. Plant-based biosensors for detecting CRISPR-mediated genome engineering. ACS Synth Biol. 2021. doi: 10.1021/acssynbio.1c00455.

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