The CRISPR gene-editing system is a marvel of modern science, but cutting strands of DNA may not be the safest or most elegant solution. Researchers at Columbia University have developed a new version called INTEGRATE that works in a similar but gentler way, using “jumping genes” that insert large sequences of DNA without breaking the strands. And they’ve now imaged the mechanism at work for the first time.
Most CRISPR systems work by scanning through the genome in search of a specific target section of DNA, cutting a piece out then inserting something new. This allows undesirable genes – such as those that cause disease – to be removed and replaced with something more beneficial. But with all that cutting and pasting, sometimes the sequence doesn’t join back together properly, and this can lead to unintended mutations.
An emerging branch of CRISPR systems takes a more gentle approach, adding new DNA sequences without cutting existing ones. Insertion of Transposable Elements by Guide RNA-Assisted Targeting (INTEGRATE) is one such system.
INTEGRATE was developed earlier this year when Columbia researchers discovered an intriguing “jumping gene” in Vibrio cholerae bacteria. As the nickname suggests, this gene was found to jump around the genome, inserting itself in different places. It does this without cutting the DNA strands, instead using another enzyme to slip itself in.
By reprogramming the RNA that guides it, the researchers were able to control where the jumping gene inserted itself. The resulting gene-editing tool, INTEGRATE, can be used to insert DNA sequences up to 10,000 bases long.
For the new study, the researchers set out to learn more about how it worked. Things move fast on the sub-microscopic stage, so it’s often difficult to see what’s going on. Enter cryo-electron microscopy. This imaging technique slows things down by flash-freezing them, then using an electron microscope to see what’s happening in very fine resolution.
The team found that INTEGRATE uses a “Cascade” or “Cas” complex, much like other CRISPR systems. This large portion scans the cell using a guide RNA, looking for a matching DNA sequence. When it finds one, it threads its own genes through TniQ “transposition” proteins, then calls in other enzymes to help change up the DNA.
Seeing this in detail confirmed a few of the team’s original hypotheses about how INTEGRATE worked. The researchers say that this new system should help make CRISPR more accurate and efficient.
"We showed in our first study how to leverage INTEGRATE for targeted DNA insertions in bacterial cells,” says Sam Sternberg, lead researcher on the study. “These new images… explain the biology with incredible molecular detail and will help us improve the system by guiding protein engineering efforts.”
The research was published in the journal Nature.
Source: Columbia University