Enzyme Case Study

The unforgettable story of vaccines is a story of triumph and controversy. The saddest part of the story is persistent ignorance and a lack of education, comingled with the personal need of some parents to explain away the problems of their children, have caused the controversy to arise. The good news is that the triumphant reality of vaccines as a whole is still the larger enduring legacy. The human papillomavirus vaccine is not an exception to this rule; in fact despite all the controversy surrounding the vaccine, it is one of medicine’s greatest lifesaving gifts to us.

With the use of CRISPR, a specific gene in the genome of a cell can be targeted and mutated to rid of the preexisting mutation. The technique works through the use of an enzyme called Cas9, which acts as the “scissors” to cut two strands of DNA at a specific location in the genome to allow for pieces of DNA to be added or removed. Another molecule of use in the process is a piece of RNA called guide RNA (gRNA). Guide RNA comprises of a small piece of pre-designed RNA sequence located within a longer RNA strand. This longer RNA strand binds to DNA and the pre-designed sequence guides the Cas9 to the correct part of the genome. This occurs for the assurance that the Cas9 enzyme cuts the right part of the genome out. The article provides specific cancers and genetic diseases and the targets for CRISPR/Cas9 that act on these mutations. Amongst the cancers, lung, thyroid, and breast cancer was mentioned. The genetic diseases mentioned were Huntington disease, Alzheimer’s and muscular

Crispr uses its protein Cas9 to precisely snip out a piece of DNA at any point within the genome and then neatly stitch the ends back together. This way of editing is effortless and has a deep appeal. This article goes in depth on how Crispr works.

Clustered regularly interspaced short palindromic repeats or CRISPR is an efficient and reliable ways to make precise, targeted changes to the genome of living cells. It is a naturally occurring defence mechanism of bacteria. The first part of the defense system is CRISPR and it just remembers parts of viruses DNA so it can recognize and defend against the virus. The second part of the defense mechanism is a set of enzymes called Cas9 or CRISPR associated proteins. Cas9 precisely cuts DNA. The CRISPR/Cas9 system was found in streptococcus pyogenes or better know as the bacteria that causes strep throat. All of this is just shortened to CRISPR. Basicly Cas9 cuts DNA and CRISPR tells it where to cut. All biologists have to do give the Cas9

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated System (CAS) is an advancement in technology that transformed a previous protein-based targeting (TALEN and Zinc Finger) process used to target and splice genes or DNA sequences. CRISPR-Cas9 was discovered in 2013 by Feng Zhang from the Broad Institute and MIT. CRISPR-Cas9 targets specific base pairs using small RNA that can be easily swapped for many different RNA targeting sites. This allowed CRISPR-Cas9 to surpass the previously used splicing methods such as, Transcription Activator-Like Effector (TALE) and zinc fingers. TALEs and zinc fingers are banded to DNA through a direct protein-DNA interaction. This was a long and tedious process that required proteins to be redesigned for each new target DNA site. CRISPR-Cas9 made the process of genome editing more efficient, effective, and precise by using RNA and proteins (“CRISPR”). CRISPR-Cas9 is a natural system that helps bacteria defend against attacking viruses known as bacteriophages. This process begins with the “CRISPR sequences [bookending] short stretches of DNA that bacteria have copied from invading phages, preserving a memory of the viruses that have attacked them in the past” (“CRISPR”). These sequences are then transcribed into short RNAs that guide the Cas proteins to match the viral sequences. The Cas proteins destroy the matching viral DNA by cutting it from the strand.

CRISPR stands for clustered regularly interspaced short palindromic repeat and the Cas9 stands for CRISPR associated nuclease nine. There are three major types of CRISPR-Cas systems and the Cas9 system is a type two system (Lundgren et al. 2015). The sgRNA used is produced from a targeting sequence (crRNA) and a Cas9 nuclease-recruiting sequence which is trans-activating CRISPR RNA (tracrRNA). The target sequence for sgRNA must be ajacent to the PAM sequence that has a base sequence of NGG or NAG (G is guanine, A is adenine and N is any nucleotide). The sgRNA

CRISPR-cas9 works by using a short guide RNA consisting of only 85 bases to precisely direct the protein Cas9 to target sites on the DNA. It then functions as an endonuclease to efficiently produce breaks in the DNA double helix.

Prior to June of 2006, Human Pappilomarius (HPV) was almost unheard of to the general public. But after FDA approval of Gardasil manufactured by Merck& Co., HPV vaccine became the headline of many articles and breaking news of many TV channels. Why? Let’s us explore it. According to World Health Organization (WHO), HPV worldwide is the most common sexual transmitted Disease (STD). There are more than 100 strains of HPV and in those 13 strains are known for causing cancer in human. HPV infection goes undetected majority of the time. HPV-infected victims mostly have no signs or symptoms or are there any tests available to detect it. The body immunity usually gets rid of the viruses on its own. However, with persistent infection, it may lead to genital warts and worrisome oro-genital cancers. With normal immunity, it takes about 15-20 years for cervical cancer to develop. Worldwide, cervical cancer is the second most frequent cancer. It is estimated more than 270,000 deaths from cervical cancer annually (1-6). In 2007, to prevent the devastation result of HPV-associated cancer, the National Advisory Committee on Immunization practices (ACIP) recommended Gardasil as a routine vaccination for girl’s ages 11 to 12 with a catch-up vaccination for those aged 13 to 26. Across the country, thereafter, state legislature attempt to make HPV vaccine mandated. As the result, controversy was generated. In this paper, we

Hereditary defects and disorders have been present throughout time, bringing about havoc on families and individuals across the globe. CRISPR technology is essentially a system in which Cas9 proteins are manipulated to apply different DNA to the cells. The Cas9 protein is existent as a protective device,

CRISPR is a new discovery, that has happened since the five to six years. This discovery is so shocking because CRISPR is a genome editing tool. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR has the ability to edit genes(adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life. The process of CRISPR is quite interesting. CRISPR first appears in viruses and when you are sick. Several studies have shown that this system can be harnessed for genome editing in mammalian cells. Cas9 is normally programmed by a dual RNA consisting of the crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid ‘guide RNA’ for Cas9 targeting. The use of a noncoding RNA guide to target DNA for site-specific cleavage promises to be significantly more straightforward than existing technologies such as ZFNs and TALENs. Using the

One of the main uses of CRISPR-Cas9 is to generate knock-out cells or animals, however modifications can be made to the Cas9 enzyme to selectively activate or repress target genes or purify specific regions of DNA. Before discussing how CRISPR works, it is important to understand its fundamental parts. There are two components of CRISPR, one being a “guide” RNA (gRNA) and the other being a non-specific CRSIPR-associated endonuclease, which is Cas9. The gRNA is synthetic RNA made up of a “scaffold” sequence for the Cas9-binding and a user-made nucleotide “spacer” sequence. The genomic DNA being addressed can be any 20-nucleotide DNA sequence as long as it meets certain conditions. This includes being unique compared to the rest of the genome and the target is present upstream of a Protospacer Adjacent Motif, or PAM. (“CRISPR/Cas9 Guide”, n.d.).

Clustered regularly interspaced short palindromic repeats (CRISPR) is a microbial adaptive immune system which utilizes an RNA guided Cas9 endonuclease to specifically target and destroy viral DNA via double stranded breaks (Cong et al., 2013; Hsu et al., 2014). Researchers discovered that they could manipulate the guide RNA (gRNA) to deliver Cas9 to a DNA sequence of interest within the human genome. This enables Cas9 to induce site specific double stranded breaks (Hsu et al., 2014). Donor DNA sequences containing fully functional genes ending in DNA that is homologous to the cleaved ends can then be incorporated into the break via homologous directed repair (HDR) by the cell’s own DNA-repair mechanism (Mali et al., 2013). In this way,

Modern genetic engineering technologies are advancing, and genetically modifying a genome is becoming a reality. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated system (CAS9) genome editing system is the most advanced in use. CRISPR can edit a genome more accurately and with higher precision than any other system. The intended use of the CRISPR/CAS9 system is to insert a gene, delete a gene, or silence a gene, leading to powerful applications (Christensen and Choy 2017a). For example, scientists plan to use this technology to change the genome and resulting phenotype in the efforts to eliminate potentially lethal diseases. One such disease is Human Immunodeficiency Virus (HIV) and Acquired

A common form of gene therapy is to take a genetically engineered virus, or a viral vector, like the Herpes simplex virus (HSV) or Adenovirus. These viruses purposes are to infect cancer cells while leaving the healthy cells in the body alone (Cross). Viruses are modified to rid the virus of the toxicity, but maintain the virus’s high gene transfer capability (El-Aneed). The genes are modified by deleting parts or all of the coding regions in the viral genome (Thomas). An RNA guided editing tool called CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats) is capable of editing multiple genes simultaneously (Xiao-Jie). CRISPR-Cas9 is a cost effective tool in gene therapy and while it is relatively new, with just being introduced for mammalian use in 2013, it is already proving to be very useful. This type of gene therapy is where problems arise with a patient’s low immune system and high risk of infections, the biological system can create an infection in the patient.

The majority of HPV infections go undetected due to an absence of inflammatory response from the host, majority of infections are asymptomatic [2]. Although most of these infections are asymptomatic eliciting no host response, this viral infection has the potential to cause detrimental disease in their host [2]. This type of viral infection was studied in the 1970’s by a scientist who dedicated his studies to understanding herpes simplex virus and its role in cervical cancers ultimately led to the discovery of HPVs [1].