An Alarm System for CRISPR Gene Editing

CRISPR gene-editing technology could help solve many of the world’s most pressing challenges. Bio-energy crops that can survive drought conditions? CRISPR may be the solution. Bacteria that turns biomass into sustainable aviation fuel? Ditto. But scientists using the technique face a problem – how can they tell if the gene edits they think they’ve made have actually taken?

Researchers at Oak Ridge National Laboratory (ORNL) have a solution. In recently published papers they detail a method of implanting biosensors that “light up” when CRISPR actions are detected. Here’s a look.

Basics of CRISPR Gene Editing

CRISPR is often described as using molecular scissors to cut and splice new material into the DNA in every cell. An alternative that may explain it better is to think in terms of replacing a word in a long document stored on a computer. The computer is told the sequence of letters to look for and what to replace them with when found. (Credit for this analogy goes to Dr. Feng Zhang at The Broad Institute of MIT and Harvard.)

In CRISPR a guide ribonucleic acid (RNA) provides the letters to be searched for and replaced. When this finds a match in the target DNA a linked protein cuts and replaces the relevant section.

Spotting the Edits

When editing a document it’s usually possible to track changes. The problem addressed by the team at ORNL is that there’s no equivalent for DNA editing. The only way to actually verify what’s been changed is through painstaking before-and-after comparison of the target genome.

The ORNL breakthrough provides a fast and efficient way of highlighting changes. In addition, it lets researchers see if any of the inserted CRISPR protein is still active. (This is a cause for concern as it could lead to unpredictable alterations of the target genome.)

How the New Technique Works

Within DNA there’s a sequence that can instruct a green fluorescent protein (GFP) to turn on. When on and subjected to ultraviolet light, green fluorescence is visible through a microscope.

The ORNL technique takes advantage of this with two tools. These are biosensor RNA that’s paired with a reporter protein. These are coded into the DNA of the organism being worked on.

The biosensor sends the CRISPR protein to the GFP sequence in the DNA. Thus if CRISPR is active the GFP will turn on. However, as the GFP signal is hard to detect ORNL replaced it with a larger reporter protein, eYGFPuv. When this turns on, excitation with UVA, (colloquially called “black light”,) yields a fluorescence that’s visible with the naked eye.

Why This Matters

CRISPR gene editing is being applied to a wide range of problems in medicine, agriculture, pathology and even pest control. However, the rate of progress is limited by the time needed to verify whether CRISPR is working, whether an organism has been modified by CRISPR and whether CRISPR is still active.

Combining CRISPR gene editing with biosensors as ORNL has demonstrated, promises to accelerate this research. It also shows the potential of biosensors for making gene-edited organisms readily detectable, both in the lab and in the field.

CRISPR has tremendous potential. The technique developed at ORNL will make it easier, faster and safer to use.

Post Author: Frida Anders