Team:CMUQ/Details
Project Details
Current CRISPR Uses:
CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9 [1]. Some potential application of this system could also be used in the treatment of different diseases, such as pulmonary, gastrointestinal, hematologic, immune system, viral, autoimmune and inflammatory diseases, and cancer [6]
1Sickle Cell Anemia
Sickle Cell Anemia is one of the most common, severe, monogenic disorders in the world. Genetic studies indicate that this condition is inherited in an autosomal recessive pattern where parents, often showing no symptoms of the condition, each contributes one copy of the mutated gene. This genetic disease is mainly caused by rs334, a single nucleotide polymorphism (SNP) located in the Hemoglobin- coding gene (HBB). Prevalence of sickle cell trait varies greatly between different regions but reaches levels as high as 40 percent in some areas of sub-Saharan Africa, eastern Saudi Arabia, and central India.
14.63% of the population are suffering from sickle cell anemia in Qatar.
Current treatment might include antibiotics, vitamins, Blood transfusions, pain-relieving medicines, other medications and possibly surgery, such as to correct vision problems or to remove a damaged spleen.
2Cystic fibrosis
Cystic fibrosis is caused by mutations in the CFTR which is responsible for coding for chloride channel into and out of cells. Chloride ions are important in cells as they regulates the movement of water in tissues (osmolarity), which is necessary for the production of thin, freely flowing mucus. The phenotype observed are difficulty breathing and coughing up mucus as a result of frequent lung infections. Other signs and symptoms may include sinus infections, poor growth, fatty stool, clubbing of the fingers and toes, and infertility in most males. There is no known statistics for the detection rate in Qatar.
Currently, there is no known cure for cystic fibrosis. Lung infections are treated with antibiotics which may be given intravenously, inhaled, or by mouth. Sometimes, the antibiotic azithromycin is used long term. Lung transplantation may be an option if lung function continues to worsen. Pancreatic enzyme replacement and fat-soluble vitamin supplementation is important, especially in the young.
Current Carrier Testing Methods for Prototype Diseases:
Although there are the traditional laboratory methods which withdraw blood to test for genetic diseases, today a variety of alternative techniques are being introduced, presenting a high competition in this field. According to the American Association of Cereal Chemist (AACC) carrier testing, today can be done by extracting genome from a body part or fluid that will then undergo PCR, DNA Sequencing, Cytogenetic Analysis, Microarrays, or Gene Expression Profiling, depending on the type of mutation searched for. The mentioned methods are observed to be time-consuming, require a lot of expertise and lab equipment. Studying sickle cell anemia as one of our prototype diseases, we came across a recent method for detecting carriers based on intrinsic characteristics and physical differences of the red blood cells as explained in this article. While such a technique could be efficient and low in cost, it remains invasive and presents biohazards. Moreover, this technique is limited to sickle cell anemia, where another non blood related genetic disease can not be detected. [5]
As for testing for Cystic Fibrosis, the most non-invasive current method is sweat test where people who have Cystic Fibrosis tend to have a much higher chloride concentration. However, to test for carriers, DNA is extracted from blood or cheek swabs and then traditional sequencing is done [6].
Project Details:
Our preliminary kit aims to provide a first step of genetic testing where if suspicious results are obtained, further and more detailed testing can be done. The biological mechanism behind this genetic test depends on the ability of the Cas12a protein to detect a specific single nucleotide polymorphism (SNP) of a gene. That is done by having a designed guide RNA (gRNA) with a fixed loop in the reaction solution where the fixed loop will help the gRNA to integrate itself within Cas12a in order to guide it to find the SNP of interest. As Cas12a utilizes gRNA and a TTTN Protospacer adjacent motif (PAM) on the DNA template to detect the SNP, upon detection, binding of the Cas12a and DNA-template occurs to form a reaction complex. This complex initiates an indiscriminate cleavage activity where single and double stranded nucleic acids get cut. Taking advantage of this characteristic, we use a single stranded DNA (ssDNA) reporter molecule which has a quencher and a fluorescent tag. When the indiscriminate cleavage activity is initiated, this ssDNA reporter molecule gets cleaved allowing the fluorescent tag to emit fluorescence as it is no longer quenched.
We endeavored to extract DNA from saliva to eliminate the biohazardous use of blood. In addition,our project design paid particular attention to the importance of genetic counseling. Genetic tests have been heavily stigmatized that people tend to either hesitate to take them or remain tense about the results, which we understand. Thus, we worked on developing an application that includes the necessary algorithm for liability calculation to prepare the patient with possible expected results. The integrated algorithm will help in saving the time and the money usually invested in finding the risk percentage of a specific disease within the normal pre-test counseling and it will psychologically prepare the patient for any type of results. Whilst the pre-counselling results will be prepared by a machine, we recommend that the device and software are to be used with people with healthcare experience in order to better deliver results. Furthermore, although we are using statistical knowledge to draw possible outcomes from the calculated liability percentage as a first step, we still recommend that the report produced by our application/software would be reviewed by a genetic counselor for deeper analysis.
References
- Adli M. (2018). The CRISPR tool kit for genome editing and beyond. Nature communications, 9(1), 1911.
- Diagnosis and Treatment of Sickle Cell Anemia, Mayo Clinic.
- Z. O. Fawzi et al(2003). Distribution of Hemoglobinopathies and Thalassemias in Qatari Nationals Seen at Hamad Hospital in Qatar. Qatar Medical Journal, Volume 2003, Issue 1, Jun 2003.
- O'Sullivan, BP; Freedman, SD (30 May 2009). "Cystic fibrosis". Lancet. 373 (9678): 1891–904.
- Rodríguez‑Rodríguez, D.R., Ramírez‑Solís, R., Garza‑Elizondo, M.A., Garza‑Rodríguez, M.D., & Barrera‑Saldaña, H.A. (2019). Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review). International Journal of Molecular Medicine, 43, 1559-1574.
- Cystic Fibrosis Diagnosis: Exams & Tests To Diagnose CF.