Difference between revisions of "Team:ASIJ Tokyo/Description"

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                         <p> &emsp;&emsp;&emsp; The exact prevalence of the ZZ genotype and associated disease is not known, but it is thought to be around 0.1% of the world population, though it varies by ethnic group. Northern Europeans, in particular, have particularly high instances of the mutation, while it is rare in Asian and African populations. The A1AT deficiency is the most common genetic cause of liver disease and the most common reason for liver transplant in children. Additionally, A1AT deficiency is often goes undiagnosed or misdiagnosed as asthma. Treatment options are also not convenient or cost-effective  - many patients require <b>weekly</b> intravenous augmentation therapy, which can cost up to <b>100,000</b> dollars a year - an astronomical amounts over a lifetime. These were also factors we considered when selecting this disease.  
 
                         <p> &emsp;&emsp;&emsp; The exact prevalence of the ZZ genotype and associated disease is not known, but it is thought to be around 0.1% of the world population, though it varies by ethnic group. Northern Europeans, in particular, have particularly high instances of the mutation, while it is rare in Asian and African populations. The A1AT deficiency is the most common genetic cause of liver disease and the most common reason for liver transplant in children. Additionally, A1AT deficiency is often goes undiagnosed or misdiagnosed as asthma. Treatment options are also not convenient or cost-effective  - many patients require <b>weekly</b> intravenous augmentation therapy, which can cost up to <b>100,000</b> dollars a year - an astronomical amounts over a lifetime. These were also factors we considered when selecting this disease.  
 
                 </p>
 
                 </p>
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<h2> CRISPR-Cas9 </h2>
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<p> CRISPR-Cas9 is a technology that enables us to edit parts of the genome by removing, adding, or altering sections of DNA. As a high school team, it was a huge learning experience to work with CRISPR gene editing, an area of great interest in the field of biotechnology. As a natural mechanism in bacteria, the CRISPR-Cas9 system is used to respond to invading viruses to cut out parts of the virus DNA, but keep a bit of it behind to serve as memory, so that future recognition and defense can be carried out. This natural bacterial system was adapted by scientists to induce change in other organisms, such as in animals. In this process, an enzyme called Cas9 acts as a pair of molecular scissors that cuts the 2 strands of DNA at a specific location when recognizing a specific sequence called a PAM sequence. gRNA, a guide RNA, guides Cas9 to the right part of the genome, allowing for specific regions to be cut, and later replaced. In our experiment, we used the Zhang CRISPR protocol to edit our point mutation, using the U6 target gRNA expression vector, with the unmutated sequence of SERPINA1, to restore SERPINA1 to its original sequence in E.coli.</p>
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     <h3> Sources </h3>
 
     <h3> Sources </h3>

Revision as of 01:47, 16 October 2018

PROJECT DESCRIPTION

Alpha-1-Antitrypsin Deficiency: What is it?

A1AT deficiency is an enzymatic liver disorder that results from a genetic mutation in the SERPINA1 gene.

    The SERPINA1 gene codes for the production of the protease Alpha-1 Antitrypsin in hepatocytes. A1AT is a protease inhibitor, and its function is mainly to protect delicate lung tissue from damage from other enzymes active in the area. Therefore, while produced in the liver, A1AT is secreted from the cells and makes its way to the lungs via the bloodstream.

   The normal version of SERPINA1 is known as the M allele and the mutation that most often results in severe A1AT deficiency creates the Z variant of SERPINA1. The SERPINA1 gene is codominant, so those with two copies of the Z allele are affected most strongly, and this is the version of the gene we aimed to correct. As you can see, the mutation is only of a single base pair, and results in glutamine replacing lysine. The relative ease by which we could use CRISPR Cas9 to fix the mutation causing this disease was also a factor in selecting this disease for study.

Mutation Anti-trypsin trying to leave the cell
What the mutation looks like The anti-trypsin trying to leave the cell

    The Z mutant version of A1AT is prone to polymerization, which renders the protein unable to be secreted from the cell producing it and builds up. However, if not polymerized and successfully secreted, the Z type A1AT is fully functional. As a result, those with the ZZ genotype usually only have 10% of normal serum levels. As only a small fraction of needed levels of A1AT are available, the medical complications related to the disease ensue. These can include liver cirrhosis and chronic obstructive pulmonary disease, especially in the form of lung emphysema, all because A1AT cannot get to the lungs to protect the tissue and instead builds up in liver cells, causing damage.This, in turn, results in recurring respiratory infections, fatigue, unintentional weight loss, and rapid heart beat upon standing. Additionally the deficiency increases one's risk of a rare type of liver cancer.

    The exact prevalence of the ZZ genotype and associated disease is not known, but it is thought to be around 0.1% of the world population, though it varies by ethnic group. Northern Europeans, in particular, have particularly high instances of the mutation, while it is rare in Asian and African populations. The A1AT deficiency is the most common genetic cause of liver disease and the most common reason for liver transplant in children. Additionally, A1AT deficiency is often goes undiagnosed or misdiagnosed as asthma. Treatment options are also not convenient or cost-effective - many patients require weekly intravenous augmentation therapy, which can cost up to 100,000 dollars a year - an astronomical amounts over a lifetime. These were also factors we considered when selecting this disease.

CRISPR-Cas9

CRISPR-Cas9 is a technology that enables us to edit parts of the genome by removing, adding, or altering sections of DNA. As a high school team, it was a huge learning experience to work with CRISPR gene editing, an area of great interest in the field of biotechnology. As a natural mechanism in bacteria, the CRISPR-Cas9 system is used to respond to invading viruses to cut out parts of the virus DNA, but keep a bit of it behind to serve as memory, so that future recognition and defense can be carried out. This natural bacterial system was adapted by scientists to induce change in other organisms, such as in animals. In this process, an enzyme called Cas9 acts as a pair of molecular scissors that cuts the 2 strands of DNA at a specific location when recognizing a specific sequence called a PAM sequence. gRNA, a guide RNA, guides Cas9 to the right part of the genome, allowing for specific regions to be cut, and later replaced. In our experiment, we used the Zhang CRISPR protocol to edit our point mutation, using the U6 target gRNA expression vector, with the unmutated sequence of SERPINA1, to restore SERPINA1 to its original sequence in E.coli.