CRISPR: SNIPPING AWAY AT DISEASE
CRISPR genome editing is one of the most exciting developments in biotechnology since its discovery a few years ago. Bacteria use this mechanism to destroy the DNA of invading viruses. Scientists subsequently discovered CRISPR’s potential for making therapeutic changes to the human genome. CRISPR therapies entered clinical trials last year. Now this amazing technology is evolving into second-generation applications such as RNA-editing and CRISPR-based diagnostics. This WEEKLY reviews how the genome editor works and explores new applications. Next week, we preview how researchers are adapting CRISPR to diagnose disease.
BAC FIGHTS BACK
CRISPR is a key immune response in bacteria. Yes, Virginia, bacteria have teeny tiny little immune systems. Like we do, these microorganisms fall prey to viral infection. (Take that, salmonella!) They’ve evolved a fascinating way to repel the invaders. In the 1980s, scientists observed a pattern in bacterial genomes: Repeating, palindromic sequences, with unique sequences—“spacers”—between repeats. They bestowed a tongue twister of a name, “clustered regularly interspaced short palindromic repeats,” on the mechanism, which we happily call CRISPR. Scientists also noticed CRISPR sequences always occur near genes that code for an enzyme that cuts DNA. This enzyme became known as Cas, or “CRISPR-associated.”
In the mid-2000s, scientists realized these spacers matched the DNA sequences of infecting viruses. The sick bacteria were stashing bits of the offending viral DNA between its own CRISPR sequences! These viral DNA snippets create a “genetic memory,” which enables the bacteria to fight back if reinfected. Here’s how:
- Viral DNA present in the spacer sequences is copied into viral RNA.
- The bacteria make the DNA-cutting enzyme Cas, which attaches to the new viral RNA.
- The resulting viral RNA/Cas complex finds its match on the invading viral DNA.
- The RNA attaches to the DNA and the Cas enzyme cuts up the foreign DNA, destroying the virus.
- Voila—“healthy” bacteria.
CRISPR TO THE PEOPLE!
In 2013, researchers adapted this defense for use in human cells. By adding a “guide RNA” and Cas enzyme to target a specific DNA sequence, scientists demonstrated the system could be used to cut human DNA in precise locations! This original Cas protein came from the Cas9 Streptococcus bacteria—hence the moniker CRISPR/Cas9.
What makes CRISPR/Cas9’s ability to cut human DNA in precise locations so cool? The protein creates double-stranded breaks (DSB) in the specified DNA sequence. Double-stranded breaks cut both strands of the DNA helix.
Think of DNA as a two-lane bridge. Now imagine an earthquake takes place causing one section to break off, falling away. DSBs can repair the damaged DNA bridge in two ways:
- Homology Directed Repair (HDR) relies on a highly similar DNA segment to repair the break. In this case, workers build a new section of the genetic bridge offsite and then helicopter it into place.
- Non-Homologous End-Joining (NHEJ) closes the gap using another strategy. Visualize workers pushing the two remaining sections of the bridge back together. NHEJ can result in a sequence error, just as sections of a repaired bridge often don’t line up properly. If the repair occurs in the middle of a gene, it typically disrupts gene function and halts the production of the matching protein.
By engineering double-stranded breaks at specific locations, scientists trigger the NHEJ or HDR cell repair pathways.
- Activating NHEJ disrupts a disease-associated gene. This prevents the production of the protein that causes the disease.
- Activating HDR fixes mutated genes by simultaneously delivering a “repair template” that contains the correct gene sequence.
Both scenarios present possible cures for different types of disease. For example, CRISPR is currently in clinical trials at Sichuan University (Sichuan, China) and University of Pennsylvania (Philadelphia, PA). Here, researchers are delivering CRISPR/Cas9 components to cancer patients’ white blood cells to disable the PD-1 gene. The PD-1 gene inhibits these immune cells. By deactivating the PD-1 gene the immune system is left intact and the patient should be able to more aggressively fight cancer. CRISPR Therapeutics (Zug, Switzerland) has begun enrolling patients for Phase I clinical trials of CRISPR to treat beta-thalassemia and sickle cell disease. Next on the research agenda come trials aimed at correcting gene sequences mutated in sickle cell disease and hereditary blindness. (Article continues below ad)
Researchers at the University of California, San Diego (UCSD), have developed RCas9, a modified version of Cas9. RCas9 targets messenger RNA (mRNA), the intermediary between DNA and proteins. Information stored in a gene (DNA) is converted into mRNA, which is then used by cells to make a protein. In other words, mRNA is a temporary copy of the permanent genetic information DNA stores.
RCas9 cuts mRNA. Scientists can target specific disease-associated mRNAs with RCas9. The UCSD team has tested RCas9 on cell-based disease models in the lab and have shown RCas9 to reduce problematic mRNAs in Huntington’s disease, myotonic dystrophy, and amyotrophic lateral sclerosis models.
Targeting disease-associated RNAs instead of genes may allow researchers to treat certain genetic diseases by getting at their root cause without permanently altering a patient’s genome. The UCSD team has launched Locana (San Diego, CA) to move this technology from the lab into the clinic.
THE LATEST MODEL OF GENETIC SCISSORS: CAS13
Cas9 is only one enzyme used in CRISPR . Its utility has inspired scientists to hunt for related proteins with slightly different applications. One of the most interesting is the Cas13 enzyme. Like RCas9, it targets mRNA.
Scientists at the Broad Institute in Cambridge, MA, modified Cas13 to edit single bases that comprise the mRNA sequence instead of just cutting mRNA. Specifically, the new and improved Cas13 changes the base adenine (A) to inosine (I). This inosine base isn’t normally found in mRNA, but our cellular enzymes recognize it as the common base guanine (G). G to A mutations play a role in Duchenne muscular dystrophy and Parkinson’s disease.
Repairing mutated mRNA may restore a patient’s normal protein function without permanently changing their genome. The Broad Institute team christened their upgraded Cas13 enzyme REPAIR, RNA Editing for Programmable A to I Replacement.
The ability to edit RNA significantly expands CRISPR’s therapeutic usefulness. Equally important, it eases potential safety concerns that could arise from making lasting changes to someone’s DNA.
Next week, we look at the flip side of therapeutic CRISPR—its use in diagnostics.
Emily Burke, PhD has worked in biopharma for 20 years, gaining science writing experience at The Scripps Research Institute and Ionis Pharmaceuticals. As a Ph.D. molecular biologist, she is passionate about advancing the public’s understanding of science. In addition to being a self-proclaimed “science geek,” she is regularly asked to speak at international scientific meetings. When not teaching and writing the WEEKLY for Biotech Primer, Dr. Burke swims with her swim club and performs regularly on the improv circuit in San Diego.