The processing of human DNA is already in clinical studies for blood diseases and eye diseases. Other … [+]
Happy DNA Day, fellow nucleic acid enthusiasts! On April 25, 2003, Congress declared National DNA Day to commemorate the date in 1953 when the double helix structure of our favorite molecule was solved. Since then, the National Institute for Human Genome Research (NHGRI) has organized an annual celebration “to provide students, teachers and the general public with an opportunity to learn about and celebrate the latest advances in genomic research and to examine how these advances are affecting their lives could. ”
We’re going to celebrate by looking around to see what CRISPR has been up to lately. I admit, I always forget what CRISPR stands for (ok, it’s “regularly grouped short palindromic repetitions in groups”) – but the official term doesn’t make exactly what it is. When we talk about “CRISPR gene editing” we are referring to a combination of an enzyme that makes a precise cut in DNA and a small piece of RNA that indicates where that cut should be made. (This is why it is often written as “CRISPR-Cas9” – Cas9 is the name of the enzyme that cuts DNA.)
Still confused? This video will help:
The CRISPR system was discovered in bacteria back in the 1980s, but was adapted for precision cutting in laboratory research in 2012. Less than a decade later, it is already being used in the clinic to treat human diseases. Last year, the Nobel Prize in Chemistry was awarded to Jennifer Doudna and Emmanuelle Charpentier, the two scientists who demonstrated the precise processing of genes with CRISPR for the first time in the laboratory.
Read on to learn about the latest developments in using CRISPR to cure disease, improve virus diagnostic tests, and develop a new type of antibiotic. Bonus: read to the end to find out how RNA is also put into action.
Gene editing for the treatment of sickle cell disease
Red blood cells that have sickle cell disease don’t look round and healthy, they take … [+]
A clinical trial will begin later this year for a new CRISPR-based therapy to repair the genetic defect that causes sickle cell disease. The study, which will be conducted by doctors from UCSF and UCLA, will enroll 9 patients to determine the safety and effectiveness of the treatment.
“Sickle cell disease is the perfect combination of all the right reasons to use CRISPR,” says Fyodor Urnov of the Innovative Genomics Institute, a joint research collaboration between the University of California, Berkeley and UC San Francisco.
For one thing, sickle cell disease is caused by a change in a single “letter” in DNA, and that’s exactly what CRISPR does: capture and change a single letter of DNA.
In addition, the need is great. Sickle cells are widespread, affecting approximately 100,000 people in the United States, mostly of African American descent, and millions worldwide. It results from a bug in a gene called beta globin, a component of hemoglobin. The disease leads to malformation of red blood cells and chronic pain, organ damage, strokes and other nasty symptoms.
To carry out the treatment, stem cells are extracted from the patient’s bone marrow. These are the cells that make red blood cells. The cells are then CRISPR-tested outside the body to fix the hemoglobin gene. Then the researchers can check the cells to make sure the processing is working as planned before they are re-infused into the body.
The beauty of this approach is that not every single cell has to be edited to cure the disease. “The pretty remarkable fact is that we don’t have to repair all of the bone marrow,” says Urnov. “There is strong evidence that repairing a smaller fragment will heal the sickle cell.”
Fast, portable diagnostic tests for viruses
Using CRISPR to cure genetic diseases is undoubtedly one of the most exciting applications, but creative inventors are thinking of many other ways that CRISPR’s precise cutting of DNA can improve biotechnology. One of them is in diagnostic tests for viral infections.
Thanks to the Covid-19 pandemic, you’ve probably heard of the “PCR test” for viral RNA. By taking a tiny starting amount of DNA or RNA and making enough copies to analyze in the laboratory, you may not know that PCR is working. It is very efficient but requires expensive equipment and trained technicians to operate.
Sherlock Biosciences has developed a rapid diagnostic test that detects genetic material from viruses in a portable room temperature format. The test is based on a CRISPR enzyme and a short piece of RNA, a so-called “reporter molecule” that glows when cut. The enzyme is called Cas13 and is a little different from Cas9. A guide RNA tells Cas13 which viral DNA sequence to look for, but if it is found, the enzyme will not cut the viral DNA. Instead, it cuts the reporter molecule.
This is how the test works. The DNA or RNA from the sample is copied into DNA using a chemical reaction called LAMP, which can be carried out in a single tube – no complex equipment required. Next, Cas13 and the reporter molecule are added to the mixture. When Cas13 hits the viral DNA sequence it starts cutting the reporter molecule which then glows and creates a nice, easy to interpret positive test. However, if the viral sequence is absent, Cas13 does nothing. No cut, no glow, negative test.
Sherlock already has a quick point of care test for Covid-19 on the market and is working on flu tests. After all, the hope is to develop tests for dangerous viruses like Ebola. Such tests could be of great benefit in areas where access to high-tech laboratory equipment is difficult.
A new type of antibiotic
Antibiotic resistance is emerging as one of the greatest health challenges of our time as bacteria develop ways to survive the toxins we throw at them. The problem will not be solved simply by improving the drugs we have. It requires entirely new ways to kill disease-causing bacteria.
The other big problem is that antibiotics can be indiscriminate. Believe it or not, your body contains more bacterial cells than human cells, and most of those bacteria are doing you a favor. There are many “good” bacteria in our digestive system that help us process nutrients and possibly even fight off various diseases. Taking antibiotics can kill the good with the bad and disrupt the whole system.
A phage is a virus that infects bacteria. Locus develops phages to transport Cas3 enzymes that … [+]
Locus Biosciences is developing a high-precision antibiotic that uses viruses called “phages” to deliver CRISPR enzymes only to bacterial cells. This combination ensures extremely precise killing of only the bacteria causing the disease without collateral damage.
“We build drugs that selectively remove bacterial species or strains without touching anything else,” said Paul Garofalo, President and CEO of Locus. The reason they are so selective is that phage target bacteria very specifically. Locus collects samples from around the world – soil samples, wastewater treatment samples, and other places where bacteria live – and examines them for phages that infect common drug-resistant bacteria that plague humans.
So far they have compiled a collection of more than 6,000 different phages. Each drug contains 3 to 6 phages that work together for maximum efficiency. Locus develops the viruses to smuggle the CRISPR enzyme Cas3 into the cell. While Cas9 has a “molecular scissors” action, Cas3 chews DNA like Pac-Man. “It swallows this one strand back and permanently destroys the reproductive capacity of this cell,” says Garofalo.
Locus recently completed a successful clinical trial demonstrating the safety of its drug for the treatment of recurrent urinary tract infections caused by E. coli. During the trial, they found that 28% of the bacteria, far more than the expected 5%, showed resistance to multiple drugs.
“Antibiotics are less effective every year,” says Garofalo. “There are no antibiotics that do not increase resistance. We are safe in a race. ”
ADAR: Precision Machining of RNA
After all, we cannot complete the DNA tag without a cameo of RNA. While Locus uses CRISPR, but not for gene editing, Korro Bio does gene editing – but not for CRISPR. They are co-opting a system called ADAR to precisely manipulate RNA.
The DNA processing changes the genome permanently and once, but the RNA processing makes a temporary correction. This can be a good thing: it allows the dosage to be adjusted, and treatment can be stopped if problems arise. ADAR stands for “adenosine deaminase that acts on RNA”, and everything that means that it is an enzyme (a deaminase) that makes a chemical change to a certain “letter” of RNA (adenosine). It doesn’t even cut the RNA. This chemical change converts the adenosine (A) to another letter, inosine (I), which changes the protein made from this RNA.
Human cells already have ADARs, so unlike CRISPR, there is no problem releasing an external enzyme into the body. Korro’s goal is to find a way to conjure the ADARs in places in the RNA where that A-to-I change fixes a disease-causing mutation. To do this, they form short synthetic RNA strands, so-called oligos, which match the RNA strand to be fixed. Once in the cell, the oligo pairs with the native RNA, and the resulting double-stranded RNA attracts ADAR to change the erroneous letter.
“It’s not a permanent edit, so it may be safer,” said Ram Aiyar, CEO of Korro. “You can switch it off. If we stop dosing, there is no processing.” This is because RNA does not hang around in the cell for very long: It is a temporary messenger molecule. Like secret commands in a spy film, it is read and then destroyed .
Korro targets disease genes in the liver, eye, and nervous system. They haven’t started human trials, but in animal studies they have successfully corrected an abnormal protein that causes liver disease. But it is in the nervous system where Aiyar suggests that RNA editing could meet the greatest need. “Some of these CRISPR systems were difficult to use in the CNS, but delivery of an oligo was not,” he says.
CRISPR and other gene editing technologies are rapidly changing medicine. How these efforts will play out, and what still unexplored paths might await us, should make this a very interesting decade for genetic medicine.