ELISA Lab Analysis

Genome editing is a huge leap forward in science and medicine. Because of recent advances in technology, the study of genes and induced ‘point’ mutations have led to the discovery and advancement of methods previously used in order to mutate genes. The development of Clusters of Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR associated system 9 protein (Cas9) technology is a hugely significant leap forward as this is a tool that could potentially be used for the research into and hopefully the treatment of a range of medical conditions that are genetically related. Cystic fibrosis (Schwank, G. et al, 2013), haemophilia and sickle cell disease are an example of some of the conditions that have the

CRISPR Cas-9 is a system that changes genes and shows a further promise for treatment for Duchenne Muscular Dystrophy (DMD). Doing this will hopefully avoid ethical dilemmas. DMD effects nearly 1 in 5,600-7,700 people between the ages of 5 and 24 for males in the U. S. CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats”. These are DNA segments found in microorganisms that repeat itself in open spaces. CRISPR attacks any virus that comes into your body and cuts the strand of virus on the DNA out. Researchers have tried to cure DMD in mice by getting therapy through the back of the eye, one of the ways CRISPR therapy works, which seems to work best.

The advantage of both studies is that both method have tested on in vivo and in vitro, which allows the future studies to work on the development of ex vivo gene correction so that a better treatment for DMD could be generated. However, both studies still have some defects relatively. For example, the protocol of the Yong et al. study was unclear, which is significant for DMD patient as the misleading protocol could be fetal to the patients. Although Lee et al. mentioned that CRISPR-Gold did not cause any inflammation or weight loss after multiple injection, it was not sufficient to suggest that the

There are many highly diverse Cas (CRISPR associated) proteins but only one that is mainly used, which is Cas9.1 Which Cas9 derivative is found on the gRNA that contains plasmid, which are associated with CRISPRs type II in the bacterial immune system. Cas9 is an important asset to a type II CRISPR system mechanism that is required for gene silencing. Cas9 protein engages in the processing of crRNA and is also responsible for eliminating the target DNA in the type II CRISPR system.1 Cas9 function as an RNA guide for the DNA endonuclease enzyme that is involved with CRISPR adaptive immune system.

genetic analysis to study the role of the Smyd1b gene in the skeletal and cardiac muscular

Even though it was discovered in 1987 by a research team at Osaka University, the true power of CRISPR, which functions as an adaptive bacteria immune defense, was not realized until the completion of the Human Genome Project in the early 2000’s (Carroll and Zhou 63-64). There are only two components of CRISPR, a guide RNA sequence (gRNA) and a Cas9 endonuclease protein. When the gRNA binds to its matching template DNA sequence, the Cas9 protein cuts the template DNA sequence resulting in the inactivation of that specific gene (Hille and Charpentier 3). In addition, a mutant gene can be replaced by adding another piece of DNA with the desired sequence, which will bind to the cut DNA template strand and become incorporated into the host

A research team at the University of Texas Southwestern medical centre have stated that by using the CRISPR-cas9 system they can target the disease within mice, and reattach fibres, therefore curing the disease. The mice after the CRISPR-Cas9 enzyme was added, were able to generate more force, grip stronger and even resist eccentric contraction and reduced blood levels of Creatine Kinase. However out of all these points the most

CRISPR-Cas9, a genome editing instrument, moves to change the field of biology forever. CRISPR was first observed as an innate defense mechanism used by bacteria. After years of development, scientists have been able to construct their own RNA that guides the CRISPR-Cas9. This allows them to control the behavior of the CRISPR-Cas9. What this could mean for the future is overwhelming.

These mice, a severe model from the Arthur Burghes lab at Ohio State University, are either stillborn or die on average at approximately postnatal day 5 with approximately 40% loss of motor neurons, representing the severe type I SMA phenotype. As is seen in human patients, mice with higher copy numbers (more than eight) of the SMN2 transgene were phenotypically rescued, once again demonstrating that the SMA phenotype could be modified by the number of copies of SMN2 (Monani et al., 2000). A similar strategy was used to generate SMA mice in the Hung Li lab in Taiwan (Hsieh-Li et al., 2000). In contrast to Burghes severe model, the Hung Li SMA mice showed a range of phenotypic severity within the same litter, from severe to moderate. Further studies indicated that this variation in phenotypic severity was likely due to hemi or homozygous transmission of the transgenic human SMN2 allele on the background of heterozygous deletion of the Smn1 endogenous allele, thereby producing mice expressing varying copy numbers of the transgene (Gogliotti et al., 2010). Building on the development of the Burghes severe model, mice expressing a SMN7 transgene in addition to full length SMN2 were also produced (Smn1-/-;SMN2tg/tg;SMN7tg/tg). Introduction of the SMN7 allele extended the life span of affected mice from approximately five days to approximately thirteen days (Le et al., 2005) and indicated that SMN produced from the SMN2 locus could be a valid therapeutic target. To date, the two most commonly used mouse models are the Smn1-/-;SMN2tg/tg;SMN7tg/tg (SMA7 mice) and the Smn1hung-/-;SMN2Hungtg/tg (Hung-Li SMA mice), which both have an average survival of approximately 13 days and recapitulate the clinical manifestations and neuropathology of intermediate type II SMA

However there is some uncertainty to the effectiveness of this treatment (Joulia-Ekaza and Cabello 2007). MSTN has been seen as a short term (9 months) treatment for diseases in mice but, long-term effects are not yet known (Joulia-Ekaza and Cabello 2007).

CRISPR Cas9 is a gene editing tool that can be used to edit, delete, and change parts of the genome. What makes CRISPR Cas9 different from other gene editing techniques such as Zinc Finger Nuclease (ZFN) or Transcription Activator-Like Effector Nucleases (TALEN) is its targeting efficiency ability. For example, ZFNs and TALENs could only reach targeting efficiencies from 1% to 50% in human cells. On the other hand, CRISPR Cas9 had a much higher rate at greater than 70% in Zebrafish and plants. (Reis, 2014). CRISPR Cas9 is still being heavily researched by scientists today, because although many advancements have been made since its discovery in 1993, it is far from ready to be used commonly. Successful studies and experiments with this gene

RESULTS. CRISPR-mediated genome editing was able to cut out the mutated exon 23 found in DMD mice. Immunofluorescence was used to demonstrate that the dystrophin protein expression was indeed reestablished. Approximately 40% of dystrophin protein expression was regained. Muscle function as well as expression was also reestablished. Cardiac muscle contractility improved significantly.

The researchers were able to show that MGC1203 is involved in BBS in several ways. They noticed that this protein was the only protein to be present in two separate data sets in which they were identifying proteins that interacted with BBS genes in yeast. 3 MGC1203 was also found in similar locations as the BBS genes in tissues that are often affected by the disorder. They saw that of a group of patients with BBS, 6.2 percent had the 430T variation as compared to only 1.4 percent in the control group, showing a strong correlation between BBS and the 430T variant of MGC1203. They then discovered that five patients had two mutations on a locus for BBS, and also found one parent that was homozygous for 430T that was unaffected. This showed that MGC1203 mutations do not cause the BBS disorder, but show that the 430T mutation influences BBS genes. Ultimately, the researchers concluded that slight mutations to this protein can contribute to the BBS disorder epistatically, by modifying BBS genes in patients that already have a genetic background for the disorder. The 430T allele will change the use of a splice junction, resulting in improper use of exons. This that leads to a loss in a message sent by MGC1203, modifying the penetrance and expressivity of BBS genes. These interactions have a strong contribution in the severity of phenotypes between various BBS

Three types of CRISPR mechanisms, the most studied type II, have been described. In Type II, DNA is infected from viruses or plasmids, subdivided into small pieces, and incorporated into a CRISPR locus between a series of short repeats (about 20 bps). The loci are transcribed

Neurological disorders often occur due to failure of proper brain development or appropriate maintenance of neuronal circuits. CRISPR/Cas9 is now used extensively to genetically alter the genomes of various species. The ability of CRISPR/Cas9 to remove DNA sequences and correct DNA mutations opens a new route to treat genetic diseases that are caused by DNA mutations. In this assignment, I’ll try to discuss how to apply CRISPR/Cas9 to establish animal models of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s disease (HD), and to treat these disorders that are caused by several of genetic mutations.