The Potential of CRISPR

  Abstract:

The newly developed technique, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is based on the bacterial “immune system”. CRISPR-Cas9 system was discovered in the 1970s in the E.Coli bacteria. This system is naturally found in the bacteria as an anti-infection mechanism. If an analogy is to be drawn, the CRISPR-Cas9 system can be compared to the antibodies in humans. When the bacteria sense a viral invasion, it targets the viral DNA and creates strands of this targeted DNA. These fragments are known as the “CRISPR fragments” and are stored as a memory when there is an invasion by the same virus. If there is an invasion by the same virus, the CRISPR fragments produce RNA which later targets the viral DNA. The Cas9 enzyme functions to destroy the viral DNA. Similarly, in the lab, a biologist can target a specific sequence of the gene and use the CRISPR “scissors” to cut the DNA at a specific site. Scientists aim to apply this technique for cancer cures, disease prevention and as a treatment to the genetic diseases. If applied properly, this technique can be useful for research and disease prevention. However, there are many ethical dilemmas. Although this is a useful technology to treat diseases, the real question is whether or not the biologists are “playing Gods” by editing and modifying the genes. These days it is possible to modify or enhance the genetics of an unborn child through the application of CRISPR. Parents can choose the appropriate genes which will help their unborn child and can also delete the genes with a mutation. This can prove helpful for the unborn child to avoid having a genetic disease. However, choosing genes that can enhance the potential of that particular child can result in unfair advantages to that child. Genetically modified children can have exceptional abilities which might be uncommon in other children. Also is it ethical to change the genes of a child without his or her consent? This remains a common debate in eugenics which involves the modification of genes of a particular person. But this is definitely a valuable technology that can serve as a solution to the different genetic diseases. 

Introduction:

Genome editing is a method utilized by the biologists to modify the genetics in order to change the traits of that particular organism. The biologists can also apply the technique of genome editing to delete certain genes that can cause diseases in an organism. This deleted gene can be replaced with another gene. In order to achieve this, the recently developed technology which is known as CRISPR is widely employed. CRISPR has the potential to cure diseases including cancer, cystic fibrosis, blood disorders, blindness, AIDS, Muscular Dystrophy, and Huntington’s Disease. While editing the genome through CRISPR, an enzyme called Cas9 is used as a “tool” that cuts the DNA at a specific location for adding or deleting the fragments. Currently, the Cas9 enzyme is widely accepted, the Cas13 enzyme is also exhibiting that some potential benefits. Although CRISPR has promising results of curing certain disease, there are ethical questions which need to be considered. This blog summarizes various researches conducted that address the potential of CRISPR. This blog also includes the various ethical debates around the utilization of CRISPR. 

The History of CRISPR:

 The idea of CRISPR was first discovered in 1987 in E.Coli, by the scientists who were analyzing the role of the genes in phosphate metabolism. In 1993, CRISPR segments were discovered in the archaea, single-celled organisms, Haloferax Mediterranei. In the initial years of the 2000s, the similarity of these fragments to that of the bacteriophages, archaeal viruses and plasmids suggested that the CRISPR can be a part of the immune system of bacteria and archaea. Once this was discovered, scientists were eager to research the mechanism of genetic elements which destroys the other genetic element. The papers published by scientists suggested that the CRISPR/Cas “cut” the invading DNA utilizing the information from the CRISPR spacers. Spacers are inserted into the CRISPR arrays and then utilized as the “guides” to determine and inactivate the invading DNA. Certain RNA fragments, crRNA, and tracrRNA, which are produced by the virus-resistant bacteria from different locations are required for cleaving the DNA. These fragments can guide the Cas9 enzyme which later cuts the DNA. The scientists later combined the RNA fragments and tagged them as “guide RNAs”. This proved to be significant research as editing genomes was now available. 

CRISPR potential as a cancer cure:

With the newly developed technique, CRISPR, there is a possibility of curing cancer without painful chemotherapy. The possibility of curing CRISPR was tested for the first time in China. The T-cells of the lung cancer patient were obtained. The PD-1 genes¹ were “switched off” with the help of CRISPR. PD1, “Programmed Cell Death Protein” produces signals which turn off the immune response and hence prevent the damage of the healthy tissues by the T-Cells. In cancer cells, this checkpoint is disabled and cancer grows without any check. The scientists in China hoped that knocking PD1 off can help the T-Cells to identify cancer and destroy the cancerous cells. When the patients were monitored for 6 months, the patients were doing fine. The first T-Cell injections were successful. The phase two trials are currently being held. This involves the trials to be tested on esophageal cancer patients. In order to implement CRISPR, further research still needs to be done.

CRISPR potential for genetic diseases cure:

CRISPR can be a technique to cure genetic diseases while the fetus is still in the uterus. Scientists are aiming to prevent the inheritance of genetic mutations through CRISPR. In order to do this, scientists inject an RNA sequence in the nucleolus of a stem cell or a zygote. This RNA sequence guides the Cas9 enzyme which leads to the desired DNA sequence. The Cas9 enzyme then “nicks or cuts” the sequence. Introducing an appropriate sequence to the DNA can result in the deletion of the mutation. This indeed is important research that might prevent genetic diseases in the future. In fact, a Netherland based stem cell research scientist, Hans Clevers, was successful in knocking off the cystic fibrosis gene in human stem cells (Grens, 2013). The target of this team was the Cystic Fibrosis transmembrane conductor receptor. Intestinal cells from two pediatric patients who were homologous for the gene were obtained and extracted. A mutation in this receptor results in Cystic Fibrosis. A mutation in this receptor is also responsible for an accumulation of mucous fluid in the pulmonary and gastrointestinal cavities, thus, causing cystic fibrosis. Introducing a donor plasmid served as a replacement sequence in the mutated allele. The cells which were isolated and cultured were proven to have a non-mutated delta F508², which is a common mutation causing cystic fibrosis. Along with Cystic Fibrosis, Duchenne Muscular Dystrophy is a genetic disease that affects 1 in 3500 male births (“About Duchenne Muscular Dystrophy,” 2013). This disease is caused by a mutation or alteration of the DMD gene, the largest known human gene, which is responsible for the production of the protein Dystrophin. This protein is important for the strengthening and protection of muscle fiber. Due to the absence of this essential protein, the muscles of the lower limbs start getting weak which eventually progresses to the entire body. Thus causing weakness and loss of function in the muscles. As this is an X-linked recessive disorder³, this disease is found in the males. Various labs have utilized the CRISPR technique to delete the mutations on exon 23⁴ in the mice which causes muscular dystrophy. After deleting the mutation, the scientists reported the production of the dystrophin fibers. They also reported an improvement in grip strength, force generation, and decreased fibrosis. Thus, CRISPR is a promising cure for Muscular Dystrophy. Similarly, the researchers at the Columbia University Medical Center and the University of Iowa have employed CRISPR to repair the gene mutation which causes Retinitis Pigmentosa, a genetic condition that affects 1.5 million cases(“CRISPR Used to Repair Blindness-causing Genetic Defect in Patient-derived Stem Cells,” 2019). The researchers isolated skin cells belonging to the patient, in order to produce stem cells. After creating the stem cells, the researchers implemented CRISPR to delete the faults in the RPGR Gene ORF Region⁵. Because of the repetitive sequence and length of this gene, this gene remains a challenge to modify. However, the scientists were successful in editing this disease-causing gene. The next task for the scientists was to convert the induced stem cells to the retinal cells. This study suggested the importance of CRISPR to heal other diseases related to photoreceptor degeneration (Bassuk, Zheng, Li, Tsang, & Mahajan, 2016). 

The potential of CRISPR in tissue engineering:

Tissue engineering is a developing field, where scientists develop a polymer scaffold that is implanted in the human body to replace the damaged tissue. For example, the common treatment for coronary heart disease is to take autologous arteries or veins and place it in the damaged site of the coronary disease. There are many advantages of implanting the autologous blood vessel including the

reduction in the risk of an immune response. However, there is an increased risk of infection at the site the vessels were extracted. The most common infection type is the Great Saphenous Vein Harvest Site infection. This kind of infection is diagnosed when the great saphenous vein is used as a coronary artery implant. If the mammary artery is used as an implant, in some cases, it can result in the resistance in the blood flow which can ultimately cause a heart attack. Hence, a solution to these problems is introduced by biomedical engineers. This solution is Tissue Engineering. In tissue engineering, a biodegradable polymer like PGA, PLA can be used as a scaffold to implant as a graft. These scaffolds are covered with autologous stem cells to facilitate the growth at the site of damage. The biggest challenge to this solution is the stem cell differentiation. However, with CRISPR stem cell differentiation can be possible through gene editing. 

The Potential of CRISPR to cure AIDS:

 AIDS is one of the leading diseases worldwide with more than 35 million people affected (Ophinni, Inoue, Kotaki, & Kameoka, 2018). This is caused by the Human Immunodeficiency Virus or HIV which destroys the human immune system. This virus destroys the T helper cells, a type of white blood cell which is a part of the immune system and replicates itself in the destroyed T Helper cells, thus preventing an individual’s immune system to fight off any other infections. In order to cure AIDS, researchers adopted CRISPR which resulted in some promising outcomes. The researchers targeted the Tat and Rev genes, which are HIV regulatory genes, through the use of guide RNAs⁶. Most importantly, the CRISPR treatments showed no signs of decreased cellular functions or off-target gene knockouts. This in-vivo research demonstrates a potential treatment for AIDS. 

The Potential to cure Blood Disorders:

Due to a mutation or an alteration in the Beta Thalassemia gene( responsible for producing hemoglobin) deformed shape of the Red Blood Cell is produced. Hemoglobin is crucial for transporting oxygen throughout the blood. A mutation in the Beta Thalassemia gene results in a faulty shape in the blood cells. The “sickle shape” of the RBCs causes clumping in the blood vessels. This can cause a lot of problems including reduced blood circulation and much worse: organ failure. Thus, many researchers are attempting to find a cure to “Sickle Cell Anemia”. A possible solution for curing the sickle cell anemia is turning on the gene that expresses fetal hemoglobin. Normally, the gene that produces fetal hemoglobin is turned off after birth. However, by disrupting the BCL11 gene (which is responsible for turning on the fetal hemoglobin gene), the researchers are hoping to cure sickle cell anemia. CRISPR can be used to disrupt the BCL11 gene. Researchers obtained Human Hematopoietic Stem Cells from patients suffering from Sickle Cell Anemia. The researchers later disrupted the BCL11 gene and transmitted it in the mice. It was reported that the mice had an increased level of fetal hemoglobin production and resistance to sickle cell anemia. More interestingly, Vortex Pharmaceuticals, Sangamo Therapeutics, and CRISPR Therapeutics have received FDA approval to conduct human trials to cure sickle cell anemia. 

The Potential to cure Huntington’s Disease:

Huntington’s Disease is an autosomal dominant disorder, which means that only one copy of the mutated gene can cause the disease. This disease results in the degeneration of the nerve cells in the brain which affects cognitive abilities and also results in involuntary movements. Repetition in the Huntingtin gene causes the development of toxic materials which usually leads to the damage of the neurons. Through CRISPR, Polish researchers were successful in deleting the repetitive sequence and inhibit the production of toxic materials. 

What is Cas13?

CRISPR/Cas9 system is based on the modification of the DNA where the Cas9 enzyme “cuts” the specific DNA sequence and the cell repair mechanism, Non-Homologous End Joining (NHEJ)⁶ or Homology Directed Repair (HDR)⁷, thus changing the genome. This can lead to the expression of the desired proteins in order to treat a specific disease. However, editing the genome can have undesired effects. What if a wrong sequence is targeted? The proteins expressed can result in undesired effects which might include cancer. That is why the Cas13 enzyme which is functions in knocking out the messenger RNA through the guide RNAs⁸. As RNA codes for the polypeptides (the protein chain), the protein expression can be altered in order to cure the diseases. A recent research study by Zhang Lab suggests that cancer-related protein expression can be reduced. 

The Ethical Debate around CRISPR:

With the promising technology of genome editing through CRISPR, it is now possible to enhance the genetic traits of an embryo. However, editing the genome of the embryo paves way for many ethical debates. A few questions are considered to question the ethicality of genome editing. Is it ethical to edit the genome of an embryo without its consent? Is it possible that through genome editing, the scientists are encouraging social discrimination? Is the genetically enhanced individual getting an unfavored advantage over the others? These questions certainly need to be considered for the germline genome edition. To discuss these questions, the United States National Academies of Science, Engineering, and Medicine had invited the Chinese Academy of Science and the United Kingdom’s Royal Society in 2015 to discuss the ethical guidelines for CRISPR (Brokowski & Adli, 2019). This committee decided that somatic editing is permissible for the cure of genetic diseases. Nevertheless, the genome editing for the purpose of the enhancement is not permissible. To date, these guidelines are maintained as embryo research still remains controversial.  
What are your opinions about CRISPR and the ethical debate surrounding it? Please comment below. 

Notes:

1: PD1 genes or programmed cell death protein regulates the human immune system by reducing the immune system attack and promoting tolerance for native cells by minimizing T-Cell inflammatory activity

2: delta F508 is a gene which is commonly mutated in a cystic fibrosis

3: X-Linked Recessive Disorder: These types of disorders are associated with the sex chromosome: X. As men contain one X gene and one Y gene, the mutation in the X gene is expressed. 

4: Exon 23: An exon is a segment on the RNA or DNA which contains the “instructions” for creating polypeptides or protein sequences. Therefore, a mutation in this exon causes muscular dystrophy

5: RPGR Gene ORF Region: Proteins, encoded by this gene, interact with the outer rod photoreceptors and are responsible for their sustenance. Mutation in this gene is associated with retinitis pigmentosa. 

6: Non-Homologous End Joining (NHEJ): A DNA Repair mechanism found in many organisms including bacteria and man. There are a series of proteins that join the broken DNA ends. Example of such a protein is DNA Ligase

7: Homology Directed Repair: a DNA Repair mechanism by introducing d DNA donor which is similar to the sequence of the broken DNA sequence. 

8: guide RNAs: an RNA sequence which binds to the desired sequence and thus is necessary for Cas binding

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