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1) Describe genome editing by CRISPR. Describe two applications of CRISPR from literature for providing insight into biological function.
CRISPR refers to Clustered Regularly Interspaced Short Palindromic Repeats. This is a technology that forms the base of CRISPR gene editing. This Technology that has provided the scientists the capability to change the organism’s DNA. Genome editing has been considered as one of the most essential group of technologies which provide the scintist to change sequence of an organisim’s DNA. In an experimental level, CRISPR-Cas9 was primarily adupted for naturally occuring genome editingf system within the bacterial cell. This particualr technology has allowed the genetic material to be added altered or remob=ved from its original location. Thus, it also showed different expression and alters the functions. It has been evidenced that the CRISPER-Cas9 was adupted within the bacteria from a naturraly occuring genome editing system. As per the view of Nihongaki et al. (2015), the snippetes of DNA from the invading viruises has been adupted by the bacteria and later on, it has been used as to create the DNA segments called CRISPER arrayes. Thus, it allows the bacteria in order to remember the virus. Due to this reason, if the virus attack the bacterial again, then it allows the bacteria to synthesise RNA segments from the CRISPER arreys in order to to target the DNSA of the viruses. Later on the Bacteria enzymes like Cas9 in order to cut the DNA of the virus and make them dsisabled. This is considered as the survival process of the bacteria. However, in recent days scintists are still working to identify that this particular approach is safe or not and effective for the mankind (Woo et al. 2015). According to the views of Nihongaki et al. (2015), for this CRISPR is recommended as a common genome editing tool. For this the double stranded DNA that are induced by Cas9 are either repaired by processes of non-homologous end joining that are prone to error or a process of direct repair of homology.
Applications of CRISPR in biological functions:
There are various applications of CRISPR in cancer research, they are, generation of cancer models and synergistic gene study using CRISPR-Cas9.
Identification of genome and cahnge the expression
It has been identified that by the process of genome editing by using CRISPER, it allows to alter the DNA sequence and modify it. In addition to thgis, it has also been identified that Cas-9 enzyme reacts like a molecular secciors and is able to cut the strand of DNA. Therefore, once a virus has attacked the human system, then, within the cell, Cas-9 enzyme has already been produced and therefore, if the particular virus again attacks the body, then the already present Cas-9 enzye acts like molecular scissors and cut the strands of DNA. This results into protect the body system from furthher viral attacks.
Application in the Generation of Cancer models
As per the views of Nihongaki et al. (2015), Cancers are caused due to the underlying genes. CRISPR genetic cancer models are developed efficiently and inexpensively that would be helpful in the field of medicine to treat cancer. This deals with the suppression of the cancer cells that are derived by lentivirus. This model could be used for chemotherapeutic effects that segregates the affected or targeted gene and also to provide proper immune to the human organoids. With the help of CRISPR-Cas9, various other models have also been introduced. Further, as per the words of Woo et al. (2015), these models would help in transforming the cancer genetics field. The CRISPR technology helps to recognize the gene that has been affected due to cancer virus and cuts out the targeted DNA. Further invasion of the virus is being detected by the RNA that has been attached with an enzyme. The tools works provides fast and efficient results as well as it is inexpensive in nature.
Application in haematological disorders
The tool CRISPR-Cas9 also helps in the treatment of haematological disorders such as thalassemia. Thalassemia is caused due to the induction of pluripotent stem cells and also due to mutation of beta globin genes. The CRISPR-Cas9 helps in correcting the beta globin gene which is the main cause of thalassemia. Further, according to Nihongaki et al. (2015), the Hematopoietic Cell Transformation (HCT) is identified for the treatment of malignant and non-malignant cases. The tool CRISPR-Cas9 is being utilized to genetically correct the induced pluripotent stem cells and haematological disorders have been reduced to some extent.
2) What are reporter genes? From literature, give examples of four different reporters that were used for understanding biological function. Explain how each reporter system helped the researchers to understand the biological function.
Reporter gene in molecular biology, is of a great interest among the scintists. As commented by Stephen Patrick & Kalber, (2017), reporter gene is basically used as the “tag” of another DNA sequence and thus it alters the phenotipic as well as genotipic expression of the particular DNA sequence. It has been identified that the expression of reporter gene can be easily monitored and thus, the target sequence can be easily tracked. As per the view of (), reporter gene is able to encode the protein products, which has been sensitively assesed as surrogate markers and at the time of conjugating with the genes of interest in the desired location. For experimental purposes the chimeric reporter gene has been used analyse the cis-acting promoter gene regions, that allows to regulate the gene expression as well as the signal transduction pathway that helps to convey the messeges from extra cellular part to the neucleus in order to repress or stimulate the gene transcription. In addition to this, it also controls the m-RNA sequence that helps to control the messege stability. Chloramphenicol acetyl transferase (CAT) and β-galactosidase are the best examples of reporter gene.
Reporter genes are the ones that are used for the detection and measurement of gene expression. These are the sequence of nucleic acids that helps to encode the easily examined protein. They are protein products that could be easily detectable and computable without degrading the tissue.
Examples of Reporters That Are Used For Biological Function and how each one of them helped to understand the researchers the biological function:
These are genes that are used to tag other genes that would act as a promoter and helps to identify the tagged genes. As per the words of Esland, Larrea-Alvarez & Purton (2018), there are four types of reporter genes that can be listed as beta-galactosidase gene, Luciferase, Green Fluorescent Protein (GFP) and Chloramphenicol acetyltransferase (CAT).
Luciferase reporter gene:
It is a reagent used in the laboratory that helps in the catalysation of reaction with a substrate in order to produce blue-light or yellow-green that depends on the Luciferase gene. In other words, this is basically enzymes that catalyses the oxidation of luciferin. For this, the catalysts required are ATP and Magnesium ion. In the presence of excess substrate, a light is emitted that is detected by photometer.
The reagent is more sensitive than beta-galactosidase gene and is obtained from Photinus Pyralis. It has been observed from the reaction that luciferin is oxidised to oxyluciferin that emits light at 560nm.
Green Fluorescent Protein reporter gene:
This reagent has been obtained from jellyfish Aequorea victori that contains a sequence of three amino acids; they are serine-tyrosine-glycine that is responsible for the fluorescence. As per the words of Esland, Larrea-Alvarez & Purton (2018), this is a kind of reporter gene that is used for the study of vibrant process within the cell in order to know the zygosity of transgenic plants. Moreover, it is also used in the diagnosis of tumour cells.
Beta-galactosidase reporter gene:
This reporter gene is also known as lacZ gene is used under the control of enhancer in a transgene expression cassette. According to the words of Stephen Patrick & Kalber (2017), these catalyses the hydrolysis of beta-galactosidase to respective monosaccharide’s via cleavage of the glycoside bond. The beta galactosidase enzymes are basically found in lysosomes of a living cell.
As per the words of Esland, Larrea-Alvarez & Purton (2018), it is seen that in eukaryotic cells, chloramphenicol acetyltransferase is the most commonly and widely used reporter gene. These genes resist the antibiotic chloramphenicol whenever identified. These have been introduced in the cells of mammals as they are naturally not present. This further catalyses the acetyl coenzyme A acetylation of the antibiotics. These are distributed among gram negative and gram positive bacteria that show high similarity around the active sites.
3) Explain any one whole genome sequencing technology? Provide two examples of genomics application in medicine from literature.
A whole Genome sequencing techniquie has been consided as the process of identifying the compelte DNA sequence of a particular organisim’s genome at a single time. This particular technology has been widely used in human genome project to recognise the sequence of DNA in human chromosome. this results into gathering immence knowledge regarding human biology and helps to design effective medicines to protect human lives from various diseases. As commented by Comenge et al. (2018), sequencing of genome is a medical process of identifying the order of all three billion bases of DNA. However, it cannot be figure out at a single time. In order to practice genome sequence successfully, it has been identified that the chromosomes are broken into several shorter pices of DNA. These single stranded short pices of DNA are used as templet for the enzyme DNA polymerase and with the help of this particular enzyme, it results into developing a new complementort piece of DNA (Mukhopadhyay et al. 2017).
DNA consists of four bases in a sequence of pairs such as adenine pairs with thymine, cytosine pairs up guanine. According to the investigations of Quick et al. (2016), these are the mechanism of sequence pairing of DNA molecules during the time of cell division. The DNA sequencing technology involves the observing of the DNA polymerase molecules that copy the DNA molecules with exact sequence and thus complement the other part. Thus, it helps to develop more number of DNA copies within living system. The observation is done by a fast movie microscope or a camera and also incorporating new bright colours for each base. That means each base would show up different colours. Thus, this technology provides much valuable information than any other instrument. There are various types of sequencing; one of them is “High throughput next generation sequencing”.
High throughput next generation sequencing is a development towards the progressed strategies of DNA sequencing. It has high end genome sequencing technologies and is inexpensive in nature. This further provides an increased coverage to identify the sequencing of DNA. Further, as per the observations of Mukhopadhyay et al. (2017), high throughput sequencing has various applications such as, three step procedure was incorporated, resequencing of DNA, epigenetics and so on.
Two examples of genomic applications in medicine:
Genomes have a wide variety of applications in the area of medicine. It has its applications in the field of pharmacology, oncology, any infectious disease and any undiagnosed disease.
1.DNA technologies have been used by the medical authorities in order to diagnose monogenic disorders, particularly a genetic disorder. It has helped the researchers to identify the genetic disorders among different humans. As per the words of Mukhopadhyay et al. (2017), this causes different arrangement of the gene cells that might regulate growth and cell division as well. The monogenic syndrome sometimes serves as complex disease models. This is further caused by the dominant mutations of the genes. Further these mutations also might result in degradation of cells that would result in serious conditions. By this method the medical authority would be able to identify whether the medical therapy would be effective or not. It is also used to diagnose infectious diseases such as drug resistant strains and Ebola virus. A compelete genome sequence of Dolosigranulup pigrum helps to identify the interstitial lung disease of a particular person. By identifying the genomic sequence of the cultured bacteria, it is then assembled properly by using the hierarcheal genome assembly process. This helps to develop medicines for the people who have faciliting lung diseases. In order to mitigate the lung problems of the sxervice users, it helps to improve the bacterial aduptive immune defence agaist the viruses.
2. Ebola virus disease has become an epedemic in West Africa and thus it has cause death of severalk people. It has been identified that the medical researchers are unable to design new drugs to control the growth of this particular virus and thus the death rate has been automatically increased. As commentd by Comenge et al. (2018), genome, sequencing helps to recognise the DNA sequence of a particular DNA base and thus, the drug designers can design the complementory base sequences of the DNA of he foreign particle and design appropriate drug to kill thgem. In this way, it also helps in designing drugs for Ebola virus and reduce the epidemic neture of the disease.
4) Describe how monarch butterfly whole genome sequence yielded insights into long distance migration?
For a large number of years, the North American monarch butterflies have been sighted to undergo long distance migrations of upto 4000km in order to reach the winter grounds in Central Mexico (Zhan et al. 2011). It has been enalysed that the monarch genome cxlearly stated a vertibrate like opsin, which has been rapidly followed in the insects. It has also been stated that the migrants have a higher lifespan and longetivity than non-migrants and have significantly high abdominal fat stores. It was also found that these butterflies have a significant tolerance to cold and are determined to fly south regardless of circumstances. As per Zhan et al. (2011), the butterflies reproduce during the spring and fly to the Southern United States for depositing their eggs on newly developed milkweed plants. Therefore, it can be stated that these groups of butterflies have mutated genomes leading to changes within their overall body structures as well as their drive. These butterflies tend to live in areas with a temperate climate but can withstand sheer colds due to the availability of fat stores. The fat stores operate to keep their bodies warm during the cold weather conditions. It has also been evidenced that the Silkmoth and the bustterflyies have different charectorestics such as silkmoths are olfactorycentric and butterflies vission cntric in nature. Due to alteration in genomic sequence, this two distinctive nature of insecs has occured.
A whole-genome shotgun approach was utilised in order to generate a draft genome of the monarch butterfly groups. A total of 273 Mb (Megabases) of Genome was collected in order to evaluate the mutations within their gene structures. On the other hand, 432 megabases of Bombyx genomes were collected for comparison of their genomic structures. It was found that the monarch genome had a lower percentage of repeat content within their genomes than that of the Bombyx variant.
In order to understand the lepidopteran proteomes of the Monarch and Bombyx, with regards to various other insect groups, twelve bugs and two mammalian species were sampled for the study. It was found that the monarch butterfly groups had a total of 3,138 (18.6%) single-copy genes and 2,514 (14.9%) many-to-many universal genomes groups. On the other hand, the Bombyx variant was stated to have 20.4% and 15.9% of single-copy and many-to-many universal genomes respectively (Zhan et al. 2011). It was found that the monarch butterflies comprised of a center arrangement of proteins with saved capacities. These saved proteins help the butterfly groups to gain strength and determination in migrating such huge distances. The proteins are broken down into amino acids upon utilisation and the protein contents are restocked after they reach their destination in central Mexico.
It can be stated that these butterfly groups require a temperate climate and thus travel to Mexico during fall due to significant cold in the States during winters. The winters in Mexico are not as cold as that of USA and thus allow the monarch butterflies to survive in the winters. However, with the rise of spring season, Mexico starts gaining heat at a significant rate and thus the butterflies travel to cooler areas such as South American countries like Peru.
Another major reason for changes within genome structures involve the hybridisation or cross breeding between different types of butterflies. Hybridization or interbreeding between species has the potential to impact adjustment and speciation in a large number of ways (Zhang et al. 2016). Hybridization can prompt versatile introgression by transmitting advantageous alleles between species thus contributed to a tougher genome conditions which allow survival in extreme conditions.
The Heliconius butterfly groups are stated to be involved in these hybridization processes thus contributing to an increased growth and development of new body structure, better abilities for survival and increased lifespan. As stated by Zhang et al. (2016), the advancement of mimicry in Heliconius has occurred due to introgression between related species of butterflies. However, there is no specific information regarding whether the monarch butterflies utilise the same process for the development of these changes and mutations. A lack of research regarding the fact contributes to the unavailability of confirmation. However, their involvement in similar interbreeding processes, can be confirmed as the cause of bodily changes.
5) What is siRNA? Provide two examples from literature how siRNA regulates gene transcription.
siRNA can be stated as a synthetic RNA duplex which involves small interfering RNA sequences effective in short-term silencing of protein coding genomes. This process can be used in the field of mRNA degradation and the experiments are conducted in short timeframes of 2 to 4 days. Small siRNAs are considered as the double stranded Dna that has been transfected into RISC. It then helps in elict gene slicing with the process of buinding with the help of appropriate complementarity to a single standred m-RNA sequence. The siRNA involves the use of a sense stand (passenger strand) and anti-sense strand (guide strand). Solid-phase chemical synthesis processes are used in order to generate highly effective synthetic siRNA sequences. These sequences are delivered to various cells using cationic lipids, polymer based re-agents and chemical modifications to the duplex present within the cell.
As per the views of Ohama et al. (2017), it can be mentioned that more than 20-30 variations of RNA molecules have emerged as some of the critical regulators of eukaryotic genomes. Hence, it can be stated that, it has highly transformed the field of Molecular biology. However, it can be stated that major advancements have been made within the Field of Discovering small nucleotides. In compliance to the aforesaid discussions, it can be concluded that major advancements have been made within the field of siRNA. In simple terms, it can be stated that, this type of RNA has been observed to be Present within the eukaryotes. In the process of gne transcription, the si-RNAs forms self reniforcing epigenetic loops. This process has been made by DNA and histone methylation process.
SiRNA mediated gene inhibition can be reflected to have become one of the most advanced tools that can be used for Vitro Analysis. On the contrary Holoch & Moazed (2015), have suggested that, in ViVO gene silencing siRNA have been observed to play a critical role in ensuring target validation. Hence, it can be inferred that, it is one of the first and the foremost techniques that can be used for designing as well as designing siRNA based therapeutics. These have also been observed to create a breakthrough in the field of RNA science.
In accordance with the inferences that have been gathered from a study by Ohama et al. (2017), it can be stated that siRNA have two different types of strands; an antisense strand and a sense strand. Each one of them has been observed to have a significance of their own. In compliance to this, it can be stated that synthetic siRNA are the most commonly developed strands used within the development of Solid phase chemical synthesis methods. The main reason for using these chemically synthesized methods is to ensure that more stabilized strands of siRNA have been developed. Moreover, it can be stated that developing synthetic siRNA strands are more cost effective accordingly.
In compliance to the processes that can be used for gene silencing, it can be mentioned that transcriptional gene silencing (TGS) is one of the most common as well as a distinct form of RNA interference or gene silencing. In compliance to this, it has been observed that TGS results in long terms as well as stable epigenetic modifications to gene modifications. In it, the epigenetic modifications that has been incorporated inside the genes can be Transferred easily top the daughter cells during cell division. However, it can be stated that there are distinct overlap between the proteins involved takes place accordingly.
In compliance to the aforesaid discussions, it can be mentioned that siRNA can be transacted to the cells with the help of proper strands. Hence, it can be inferred that the double small strands acts as guides that can be used to load in the RISC. It is usually done with the help of cationic lipid in most of the cases. Whereas, on the contrary, in alternative cases, transacted agents gathered from polymer based substances are being used accordingly. From the aforesaid statements, it can be inferred that siRNA is an important pathway that can be used to regulate gene expressions and genome silencing accordingly.
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