Animal Genetic Modification Essay

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

Humans have been genetically engineering organisms for nearly 10,000 years using traditional methods of modification—among these methods include selective breeding and crossbreeding. Though effective, these methods were unreliable and were only able to change certain traits. A lack of control over our genetic material proved to be a clear hindrance to our species; when harnessed, advancements in other fields of knowledge would be immeasurable. Once seen as an impossible task, scientists have been able to exploit genes and take control of them. CRISPR-Cas9 is a system that allows scientists to cleave off sections of DNA and artificially modify them by inserting a mutation into the place of the old DNA. This is exceptionally precise, whilst

From the science community perspective, the CRISPR-Cas system could reduce or even eliminate many of the difficulties researchers face when gene editing such as cost, duration and accuracy. Prior to CRISPR-Cas, gene editing was performed in “big labs” with experts

For many years biomedical researchers like myself have been trying to create more proactive ways to amend the genome for living cells. In more recent fieldwork studies there has been a new state of the art instrument based on bacterial CRISP in close works with protein 9 often referred to as CAS9 from the streptococcus progenies have possibly unlocked new data. The CRISP/CAS9 tries to manipulate the function of the gene using homologous recombination and RNA interference, but is set back because it can only provide short term restriction of the genes function and it’s iffy off- target effects.

Every few years, advancements in technology alter the way scientists do their work. Recently CRISPR-Cas9, a RNA useful for working organisms in the animal kingdom has proven itself beneficial on a gene-editing platform. After performing many abortive attempts to manipulate gene function, including homologous recombination and RNA interference, scientists have finally had a breakthrough with CRISPR-Cas9.

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

Even though the CRISPR/Cas9 system is not a new phenomenon, the novelty of this research lies in the intervention of gene-editing in intraocular pathological angiogenesis. Abnormal angiogenesis is characteristic of diseases associated with blindness affecting millions of people of all ages. Since CRISPR/Cas9 technology produces a permanent genetic alteration, it

A simple but effective method is correctly designing gRNAs. Ideally, the target sequence must be a unique and nearly perfect homology to the gRNA with as little to no homology at other points within the gene sequence. Another potential strategy involves reducing the concentration of gRNA and Cas9 that are expressed in cells. However, this strategy is considered inconclusive due to inconsistent reporting. Some cell lines showed a large decrease in rates of off-target binding while others showed no effect. An increasingly promising method for improving specificity involves using a mutated nickase version of the Cas9 enzyme. This modified version contains only one catatylic domain and is used as a single nickase system capable of cutting a single strand of DNA. This results in a “nick” or single stand break rather then a DSB and is repaired by HDR. To further reduce the risk of off target binding, a double or dual nickase CRISPR system can be implemented. This system uses two nickases which bind to opposite strands of the target gene, generating a DSB. This system closely resembles the Fokl enzyme activation used in TALEN, in that off target effects are reduced because the probability of two off target nicks within the same region is very unlikely. When the DSB is made, activation of NHEJ is initiated; to further increase specificity and reduce off-target effects, the HDR mediated editing is advised for both nickase

The usage of nucleases is rapidly growing in an effort to save millions of lives worldwide; recently, gene editing tool was used to treat little girl’s cancer. Targeted genome editing is the most efficient, and effective way to treat cancer. Generally speaking CRISPR/Cas9 a powerful gene-editing tool should be considered to help the millions people that are losing their lives daily. As a matter of fact, researchers have developed three different kinds of gene-editing tools known as ZFN, TALEN, and newest and very effective CRISPR/Cas9. In the final analysis, CRISPR/Cas9 is the gene-editing tool that must be used in treatment of millions of people who are living with cancer. Recently, experiments were performed using CRISPR/Cas9, for instance editing the mushroom, modified human embryos, targeted mutagenesis, and genome editing in sea urchin embryos indicate high levels of efficiency, accuracy, and is opening door to treating deadly

RNA stands for ‘ribonucleic acid’ and it is present in all living cells. RNA’s main job is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins. RNA is similar in structure and properties to DNA. RNA, however, has only a single strand of bases. Also, rather than having the base thymine, RNA has the base uracil. Anyways, the guide RNA is composed of a miniscule pre-designed RNA sequence. This sequence is roughly 20 bases long and is located within a longer RNA framework. The scaffold part of the RNA binds to DNA and the pre-designed sequence. It ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.

Total RNA was extracted using the Trizol extraction kit (Invitrogen, Carlsbad, CA). First-Strand Synthesis System for RT-PCR (Invitrogen) was used to synthesize cDNA from 1.5 μg total RNA according to the oligo (dT) version of the protocol. Real-time PCR was performed using CFX Fast real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA). The following cycle parameters were used for all experiments: 20s at 94°C, 30s at 60°C, and 30s at 72°C for a total of 45 cycles. The relative level of mRNA for a specific gene was normalized to GAPDH levels. Table 1 shows the sequences for all primer sets used in these

Hypothesis: Ψ-mRNA, synthesized via in vitro transcription, will have enhanced stability and decreased immunogenic properties. Restriction enzymes will be used to clone the NT-3 gene (NTF3) into the pDNA expression vector known as pDNA3.1. DNA polymerase, deoxynucleotide triphosphates (dNTPs), a forward primer containing a T7 promoter, a reverse primer containing a 75 thymine tail, and the pDNA3.1 vector will be combined in a polymerase chain reaction (PCR) tube. Standard PCR techniques

In the experiment, an AAV-based CRISPR/Cas9 system was utilized for targeted gene disruption in postmitotic photoreceptors. The Cas9 coding sequence was placed in between a photoreceptor-specific human rhodopsin kinase (RK) promoter and a synthetic polyadenylation signal, and a human RNA polymerase III promoter U6 was used to drive sgRNA expression. The sgRNA vector included tdTomato expression cassette to track transduction. The RK promoter-driven tdTomato was only expressed in the photoreceptors which verified that the AAV-CRISPR/Cas9 system was specific to the photoreceptor. The sgRNA-Nrl region was sequenced, and it was found that the 98% of the changes occurred exclusively at the targeted genome site. The sequence alterations resulted in frameshifts (Yu et al., 2017).

CRISPR-cas9 allows researchers to edit part of the genome by inserting, deleting, or altering sections of the DNA sequence. The CRISPR-cas9 system consists of two key molecules: Cas9 and single-guide RNA (sgRNA). The enzyme Cas9 is ‘guided’ by sgRNA to the DNA sequences where it cuts the two strands of DNA at the specific location in the genome. Endogenous DNA repair mechanisms try to repair the breaks in both strands which then leads to indels that can cause mutation in the target gene. This process is shown in Figure 1.1. To determine whether CRISPR–Cas9 can be used to understand gene function in human preimplantation development, the POU5F1 gene that encodes the development regulator OCT4 was selected as the target. It was predicted that

Geneticist, Jay Shedure, approached cell lineage tracing with the vision to follow mutated embryonic cells through their development. Shedure utilized the CRISPR-Cas9 as part of his methods to follow mutations he induced in zebrafish. CRISPR uses the Cas9 enzyme to modify specific DNA sequences with the help of an RNA molecule which finds the desired sequence of DNA to be edited. Shedure used CRISPR-Cas9 to insert mutations in specific regions of two zebrafish embryonic genomes and then sequenced the same regions in an adult zebrafish. With the embryonic mutated genomes and the sequenced regions in the adult zebrafish, a lineage was created. Unanticipated results