Sickle Cell Disease or Anemia and CRISPR-Cas9 Genome Editing
Genome Editing and Disease
Sickle cell disease is a very unpleasant and often painful condition in which red blood cells are misshapen, stiff, and sticky. The abnormal cells may block blood vessels. Blockages can lead to tissue and organ damage and an early death. The disorder is caused by a gene mutation in a specific type of stem cell. A process known as CRISPR-Cas9 has been used to correct the mutation in stem cells placed in lab equipment. The edited cells may one day be placed in the bodies of people with sickle cell disease. The process will hopefully cure the disorder.
Many people working in molecular biology and biomedicine are excited by the CRISPR-Cas9 process. It offers the potential for huge benefits in our lives. There are some concerns about the process, however. Our genes give us our fundamental characteristics. While it's hard to imagine that anyone would object to replacing genes in order to help people with a life threatening, painful, or debilitating disease, there are worries that the new technology will be used for less benign purposes.
Living With Sickle Cell Disease
What Is Sickle Cell Disease?
Sickle cell disease is also known as SCD and sickle cell anemia. Some people use the term sickle cell anemia only for a particular variety of the disease, as described below. Patients with SCD make an abnormal form of hemoglobin due to a gene mutation. Hemoglobin is a protein in red blood cells that transports oxygen from the lungs to the body's tissues.
Normal red blood cells are round and flexible. The cells in someone with SCD are sickle-shaped, stiff, and inflexible due to the presence of the abnormal hemoglobin inside them. Normal cells can squeeze through narrow passages in the circulatory system. Sickled cells may get stuck. They sometimes collect and stick together, forming a bottleneck. The clump of cells reduces or prevents oxygen from getting to the tissue beyond the bottleneck and may cause damage to the tissue.
Sickle cell disease requires a doctor's diagnosis and treatment recommendations. Treatments vary and depend on a person's symptoms, age, and other health problems.
Possible Symptoms of SCD
Symptoms of SCD vary considerably. They depend on a person's age and the type of sickle cell disease that they have. There are some symptoms that are more common than others, however. A patient often experiences pain when sickled red blood cells block a vessel and prevent oxygen from reaching tissues. The painful episode is known as a crisis. The frequency and severity of crises is different in different people. As a person grows older, they may experience chronic pain.
Patients with SCD frequently suffer from anemia. This is a condition in which the body contains an insufficient number of red blood cells and is therefore unable to transport enough oxygen to the tissues. Sickled red blood cells live for a much shorter time than normal ones. The body may not be able to keep up with the demand for new cells. The main symptom of anemia is fatigue.
Other symptoms of SCD may include:
- an increased risk of infection due to spleen damage
- jaundice due to the presence of yellow bilirubin released by excessive red blood cell breakdown
- stroke due to the blockage of blood travelling to the brain
- acute chest syndrome (sudden breathing problems due to the presence of sickled cells in the blood vessels of the lungs)
Difference Between SCD and Sickle Cell Anemia
Sickle cell disease is a group of related disorders. It's caused by a mutation in a gene that codes for part of the hemoglobin molecule. Each of our chromosomes has a partner chromosome that contains genes for the same characteristics, so we have two copies of the hemoglobin gene. The effects of the mutated gene depend on the way in which it's altered and whether an alteration occurs in both copies of the gene or in only one.
Normal hemoglobin is also known as hemoglobin A. In certain situations, an abnormal form of the protein known as hemoglobin S causes red blood cells to become sickled. Some examples of sickle cell disease and their relationship to hemoglobin S are listed below. Other types of SCD exist in addition to the ones that are listed.
- If one hemoglobin gene codes for hemoglobin S and the other gene codes for hemoglobin A, the individual won't have sickle cell disease. The normal gene is dominant and the mutated one is recessive. The person is said to be a carrier for sickle cell trait and may pass it to their children.
- If both genes code for hemoglobin S, the person has sickle cell anemia.
- If one gene codes for hemoglobin S and the other codes for hemoglobin C, the person has sickle hemoglobin C disease.
- If one gene codes for hemoglobin S and the other codes for hemoglobin D, the person has sickle hemoglobin D disease.
A Doctor Discusses SCD
Medications and other treatments are available to treat sickle cell disease. A person may need to seek medical aid during a crisis. As the doctor in the video above says, SCD must be managed carefully because there are several symptoms associated with the disorder that are potentially life threatening. As long as this management takes place, however, the outlook for patients today is much better than it was in the past.
According to the NIH (National Institutes of Health), in the United States the predicted lifespan for SCD patients is currently forty to sixty years. In 1973 it was only fourteen years, which shows how much treatment has improved. Nevertheless, we need to find ways to increase lifespan to a normal length and to reduce or preferably eliminate crises. It would be wonderful to eliminate the disease altogether. Correcting the mutation that causes the disorder might enable us to do this.
Mutations in Hematopoietic Stem Cells
Our blood cells are made in the bone marrow, which is located inside some of our bones. The starting point for blood cell production is the hematopoietic stem cell, as shown in the illustration above. Stem cells are unspecialized, but they have the wonderful ability to produce the specialized cells that our body needs and well as new stem cells. The mutation that produces SCD is present in the hematopoietic stem cells and passed to the red blood cells, or erythrocytes. If we could give SCD patients normal stem cells, we could cure the disease.
At the moment, the only cure for SCD is a bone marrow or hematopoietic stem cell transplant using cells from someone that lacks the mutation. Unfortunately, this isn't a suitable treatment for everyone due to their age or the incompatibility of donor cells with the recipient's body. CRISPR may be able to correct the mutation in the patient's own stem cells, eliminating the problem of incompatibility.
Cell Vocabulary: DNA, Chromosomes, Genome, and Genes
DNA and Chromosomes
In order to get a basic understanding of the CRISPR process, some knowledge of cell biology is needed. Nucleic acids (DNA and RNA), the genetic code, and a specific enzyme are all involved in the process.
DNA stands for deoxyribonucleic acid. There are forty-six DNA molecules in the nucleus of each of our body cells. Each molecule is associated with a small amount of protein. The union of a DNA molecule and protein is known as a chromosome.
Genome and Genes
Our genome is the complete set of all the DNA in our cells. Most of our DNA is in the nucleus of our cells, but some is located in the mitochondria. Genes are located in DNA molecules and contain the code for making proteins. Part of each DNA molecule is non-coding, however.
Every cell is our body has almost exactly the same genes. There may be a few differences between the genes of different cells due to mutations or mistakes in cell processes.
Human Male Karyogram
Humans have forty-six chromosomes, or twenty-three pairs. The last pair are the sex chromosomes. Females have two X chromosomes, which look similar to one another. Males have one X chromesome and a shorter Y chromosome. Egg and sperm cells contain only one chromosome of each pair.
Structure of DNA and the Nature of the Genetic Code
A DNA molecule consists of two strands consisting of smaller molecules. The strands are bonded together to form a ladder-like structure. The ladder is twisted to form a double helix.
The most significant molecules in a strand of DNA as far as the genetic code is concerned are known as nitrogenous bases. There are four of these bases—adenine, thymine, cytosine, and guanine. Each base appears multiple times in the strand. The sequence of bases on one strand of the DNA forms a code that provides instructions for making proteins. The length of DNA that codes for one protein is called a gene.
The proteins that are made by cells are used in many ways. Enzymes are one type of protein and are vitally important in our body. They control the myriad of chemical reactions that keep us alive.
The "building blocks" of a DNA molecule are known as nucleotides. A nucleotide consists of a phosphate-deoxyribose group and a nitrogenous base.
Messenger RNA, Complementary Base Pairing, and Mutations
Although the code for making proteins is located in the nuclear DNA, the proteins are made outside the nucleus. DNA is unable to leave the nucleus. RNA, or ribonucleic acid, is able to leave it, however. It copies the code and transports it to the site of protein synthesis in the cell.
There are several versions of RNA. They have a similar structure to DNA but are single-stranded. The version that copies and transports information out of the nucleus during protein synthesis is known as messenger RNA. The copying process is based on the idea of complementary bases.
Complementary Base Pairing
There are two pairs of complementary bases in nucleic acids. Adenine on one strand of nucleic acid always bonds to thymine on another strand, and vice versa. The bases are said to be complementary. Similarly, cytosine on one strand always binds to guanine on another strand, and vice versa. This feature can be seen in the DNA illustration above.
The messenger RNA that leaves the nucleus contains a base sequence that is complementary to the one in DNA. The two strands of the DNA molecule temporarily separate in the region where messenger RNA is being made. Once the RNA is complete, it separates from the DNA molecule and the strands of DNA reattach.
In a mutation, the order of bases in a region of a DNA molecule is changed. As a result, the RNA that is made from the DNA will also have the wrong sequence of bases. This will in turn cause an altered protein to be made.
RNA transcribed from DNA is sometimes used for purposes other than protein synthesis. In bacterial CRISPR, RNA is used in the attack against viruses.
Bacterial Origin of CRISPR
In the 1980s, researchers noticed that several species of bacteria contained a strange pattern in their DNA. The pattern consisted of repeating sections with an identical base sequence alternating with sections with a unique sequence. They called this observation CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Today the term refers to a specific type of genome editing.
The researchers eventually discovered that the unique sections in the bacterial DNA came from viruses that had entered the bacteria. The bacteria were maintaining a record of their invaders. This enabled them to recognize the viral DNA if it appeared again and then mount an attack against it. The system is reminiscent of the action of our immune system. The process is important in bacteria because intact viral DNA takes over a bacterial cell and forces it to make and release new viruses. The bacterium is often killed as a result.
CRISPR in Bacterial Cells
The "weapon" in the bacterial attack against viruses is a set of Cas (CRISPR-associated) enzymes that cut the viral DNA into pieces, thereby preventing it from overtaking the cell. The steps in the attack are as follows.
- The viral genes in the bacterial DNA are copied into RNA (via complementary bases).
- Cas enzymes surround the RNA. The resulting structure resembles a cradle.
- The cradle travels through the bacterium.
- When the cradle encounters a virus with complementary DNA, the RNA attaches to the viral material and the Cas enzymes break it up. This process prevents the viral DNA from harming the bacterium.
The CRISPR sequence in its DNA enables the bacterium to recognize the virus. The Cas enzymes enable it to destroy the intruder.
MIT Description of Genome Editing With CRISPR-Cas9
How Does CRISPR Work in Human Cells?
CRISPR in humans follows a similar pattern to the process in bacteria. In human cells, however, the RNA and enzymes attack the cell's own DNA instead of the DNA of an invading virus.
The most common form of CRISPR at the moment involves the use of an enzyme called Cas9 and a molecule known as guide RNA. Understanding all the details of CRISPR requires an in-depth knowledge of molecular biology. The overall process as it applies to correcting mutations is as follows.
- The guide RNA contains bases that are complementary to those in the mutated (altered) region of the DNA and therefore binds to this region.
- By binding to the DNA, the RNA "guides" the molecules of the Cas9 enzyme to the correct place on the altered molecule.
- The enzyme molecules break the DNA, removing the target section.
- The cell is left to fill the gap itself with the correct nucleotides or the scientist adds a pre-made strand of nucleotides to reduce the chance of repair errors.
CRISPR and SCD: UC Berkeley Research
CRISPR-Cas9 and Sickle Cell Disease Treatment
The results of the new research into treating SCD with CRISPR are exciting. The research was performed by scientists from UC Berkeley, UC San Francisco Benioff Children's Hospital Oakland Research Institute and the University of Utah School of Medicine.
The scientists extracted hematopoietic stem cells from the blood of people with sickle cell disease. The researchers were able to correct the mutations in the stem cells by using the CRISPR process. The plan is to eventually put the edited cells into the bodies of people with SCD. The transplantation of edited cells has not yet been done, however. The researchers have to perform large scale tests with mice and lots of safety tests before they will be allowed to perform clinical trials in humans.
Adding normal stem cells to the body will be useful only if the cells stay alive. To discover whether this is possible, the researchers placed edited hematopoietic stem cells in the bodies of mice. After four months, two to four percent of the mouse stem cells that were examined were the edited version. The researchers say this percentage is likely the minimum level needed to be beneficial for humans. They want to increase the survival rate of the cells before they begin clinical trials, however.
Unlike other gene-editing methods, it (CRISPR) is cheap, quick and easy to use, and it has swept through labs around the world as a result. Researchers hope to use it to adjust human genes to eliminate diseases, create hardier plants, wipe out pathogens and much more besides.— Heidi Ledford at nature.com
Hope for the Future
Many people with SCD as well as their caregivers are probably eager to receive a transplant of genetically corrected stem cells. Scientists need to be cautious, however. Changing the DNA of a living person is a very significant event. Researchers must make sure that the altered stem cells are safe.
The researchers estimate that they will be ready for a clinical trial in humans in a few years. This may seem like a long time to patients. The wait could be very worthwhile if it enables scientists to provide both effectiveness and safety with their genome editing technique.
Questions & Answers
© 2016 Linda Crampton