Imagine having the ability to edit the mutations out of your own genes. Genetic diseases like Huntington’s, Tay-Sachs and cystic fibrosis would become a thing of the past; this ability would change the face of medicine. The potential applications of gene editing are far-reaching — and new research from Cornell might get us closer to making these applications a reality.
A recent study may have uncovered another mechanism of a new gene editing technique. Prof. Ailong Ke, molecular biology and genetics, has been leading research on the structure of Type I Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems, which have the potential to be more specific than current gene editing techniques.
CRISPR refers to short repeating DNA sequences normally found in bacterial genomes that are important in the bacterial immune system. When bacteria are infected with viruses, a small portion of viral DNA can be integrated in the bacterial DNA between these CRISPR sequences. They serve as a database of foreign invaders, allowing the bacteria to easily recognize and attack viruses. Bacteria are then able to synthesize RNA molecules made from both CRISPR and viral DNA and use this RNA as a guide to find and cut up DNA from invading virus particles.
CRISPR immunity was discovered nearly a decade ago, but its new potential applications were only discovered in the last few years. With modifications, the CRISPR immune system has been transformed into a gene editing technique that can insert or delete genes at a target region with unprecedented accuracy. The key feature of this technique is a programmed CRISPR-Cas9 system — it uses a synthesized RNA guide that can target nearly any desired DNA sequence. Once this system locates the sequence of interest, it uses the enzyme Cas9 to cut the DNA at that location. CRISPR can be applied to many different animal systems, including humans, but the full implications are still being studied. For now, the technology is primarily used in basic science research.
CRISPR technology has immense potential, but is still very new. Most research has been done on Type II CRISPR-Cas9 systems, which use a single protein, Cas9, combined with a guide RNA to target a 20 nucleotide long DNA sequence. Research done in the Ke Lab investigates Type I CRISPR systems, also known as Cascade: CRISPR associated complex for antiviral defense. For the first time, they were able to uncover the mechanism of Type I Cascade.
In their recent paper published in Nature, Robert Hayes — a postdoctoral fellow in the Ke Lab and first author of the paper — describes some the key features of Type I Cascade. Unlike Type II CRISPR systems, Type I Cascade is made up of several proteins and uses guide RNA to target a 32-35 nucleotide sequence.
“The longer RNA sequence in Cascade could potentially lead to higher specificity in targeting,” Hayes said.
The researchers also discovered that Type I Cascade has a different DNA detection mechanism. DNA in its normal state forms a double helix, often compared to a spiraling ladder. Because of the naturally uneven spacing between DNA strands, the DNA has major and minor grooves. When viewed from the side, major grooves are located where DNA backbones are farther apart, while minor grooves are the spaces where they are closer together. Type I and Type II systems must first check a short nucleotide sequence before fully unwinding the DNA to see if it is a match for the RNA. To do this, they read sequences from the grooves of the DNA.
According to Hayes, most Type II systems recognize the major groove, while Type I systems read the sequence from the minor groove, a far less stringent form of recognition.
“[This could] potentially increase the number of targets that Cascade could be programmed to interrogate” Hayes said.
Using X-ray crystallography, the team was able to capture a snapshot of Type I Cascade in action. From this, they determined the three-dimensional atomic structure of the protein complex attached to its guide RNA as it was interacting with its DNA target.
“The crystal structure gives us the necessary information to potentially engineer new Cascade variants,” Hayes said. With this structure, the team could create a new tool in gene editing.