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SARS-Cov2 and Crispr-Cas Gene Assignment

Sample guide assignment on the activity regarding elucidation of morph...
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Genetics, Lecture (BIO130.01)

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Cracking the Code for SARS-Cov2 and the Discovery of CRISPR-Cas Gene Editing Mechanism

SARS-Cov 1.) Describe the morphological structure of SARS-Cov2 virus.

  • According to studies through the use of Electron microscopy, the structure of SARS-CoV-2 is speculated to be similar to that of SARS-CoV with a virion size ranging from 70 to 90 nm (Kumar et al. 2020). The SARS-CoV-2 virus is also found to have surface viral proteins, namely, the spike glycoprotein (S) which mediates interaction with cell surface receptor ACE2, as well as, viral membrane (M) and envelope (E) viral proteins that are embedded in host membrane-derived lipid bilayer encapsulating the helical nucleocapsid comprising viral RNA (Kumar et al. 2020).

2.) In a complete statement, state the size of the SARS-Cov2 genome.

  • The size of the SARS-CoV-2 genome is found to be in the range of 26 up to 32 kb (kilobases) and is comprise of 6 to 11 open reading frames (ORFs) encoding 9680 amino acid polyproteins (Kumar et al. 2020).

3.) How does the SARS-Cov2 infect (enter) its host cell and how is it replicated inside the host cell?

  • The SARS-CoV-2 virus, like SARS-CoV, enters into its host target cells through the binding of spike protein, which exhibits 10-20 times higher affinity than that of SARS-CoV, to the angiotensin-converting enzyme 2 (ACE2) receptor for internalization and then the use of transmembrane serine proteases (TMPRSS2) for S protein priming (Kumar et al. 2020). This then results in conformational changes in the spike protein that leads to the fusion of the viral envelope (E) protein with the host cell membrane following the entry via an endosomal pathway (Kumar et al. 2020). This internalization or entry is followed by the release of the viral RNA into the host9s cytoplasm where it will undergo translation and generate replicase polyproteins ppla and pplb which is then further cleaved by the virus9 encoded proteinases into small proteins. Replication of coronavirus which involves ribosomal frame-shifting during the translation process then proceeds (Kumar et al. 2020). Through discontinuous transcription , the replication process then generates both genomic and multiple copies of subgenomic RNA

species that encode for relevant viral proteins. The virions, on the other hand, that are subsequently released out of the host9s cells via vesicles, assemble via the interaction of viral RNA and protein at the endoplasmic reticulum (ER) and the Golgi complex (Kumar et al. 2020).

4.) Give at least three potential hosts (organisms indwelt) by the SARS-Cov2 virus.

  • Since the genomic characterization of SARS-CoV-2 has shown that it is of zoonotic origin, which means that it has jumped from a non-human animal (usually vertebrate) to a human, one of its potential hosts would then be humans (Kumar et al. 2020). Moreover, since phylogenetic analysis of the SARS-CoV-2 virus revealed that it might have originated from bats or pangolins , these animals would then also be among the potential hosts of the virus.

5.) Name two protein receptors found in the cell membrane of host cells that are responsible for the entry of the SARS-Cov2 in the host cell.

  • The two protein receptors in the cell membrane of host cells that are the main determinants responsible for the entry of the SARS-CoV- virus are the angiotensin-converting enzyme 2 (ACE2) receptor and the transmembrane serine protease (TMPRSS2) (Kumar et al. 2020). The ACE2 receptor is used by the virus for internalization through the attachment of the S glycoprotein to the receptor, while TMPRSS2 serine proteases, on the other hand, is used for S protein priming that leads to conformational changes which allows for the virus to enter the cells (Kumar et al. 2020).

6.) Conceptualize a <drug= that can disrupt a specific step in the Central Dogma and how it functions to inhibit this step.

  • As obligate intracellular parasites, viruses are completely dependent upon their host cell for their replication processes (includes transcription of mRNA and translation of viral proteins). As such, it can then be concluded that to inhibit viral development, it is then needed to also inhibit the process of central dogma in the host cell (Louten 2016). In the case of the SARS-CoV-2 (COVID-19) virus, since its pathogenicity is directly related to its viral proteins, such as its spike glycoprotein (S) that exhibits almost 10-20 times higher binding affinity to the ACE2 receptor of the host cell than that of SARS-CoV; inhibiting the production of such viral proteins by disrupting the process of

collecting a sample from the parts of the body where the virus gathers, such as a person9s nose or throat in the case of SARS-CoV- virus (Jawerth 2020). Sample is then treated with several chemical solutions so as to remove substances such as proteins and fats, thus, allowing for only the RNA (a mix of the person9s own genetic material and, if present, the virus9 RNA) present in the sample to be extracted (Jawerth 2020). After extraction, the RNA is then reverse transcribed to DNA using a specific enzyme. Scientists will then add additional short fragments of DNA that are complementary to specific parts of the transcribed viral DNA so that if the virus is present in a sample, these fragments will then attach themselves to the target sections of the viral DNA (Jawerth 2020). Some of these added genetic fragments are used for building DNA strands during amplification, while the others are used for building the DNA and adding marker labels to the strands which are then used to detect the virus (Jawerth 2020). The mixture is then placed in an RT-PCR machine where it will cycle through temperatures which will trigger specific chemical reactions that will create new, identical copies of the target sections of the viral DNA (Jawerth 2020). Since the marker labels attach to the DNA strands, which release a fluorescent dye, increases as new copies of the viral DNA sections are built , the machine9s computer then tracks and presents the amount of fluorescence in the sample every cycle in real time. When a certain level of fluorescence is surpassed, confirmation of the presence of the virus, as well as, its severity is then done (the fewer the cycles, the more severe the viral infection is) (Jawerth 2020). PCR is a technique that allows a very small well-defined segment of DNA to be amplified, or multiplied many times so that there9s enough of it to be detected and analyzed. However, since viruses such as SARS-CoV- do not contain DNA but instead have RNA, the process <reverse transcription= that is an added step to RT-PCR is then done so as to convert RNA into DNA. (Hirst 2020)

(Image Source: bcgsc/news/how-bc-scientists-are-addressing- covid-19-testing-challenge ; rockedu.rockefeller/component/qpcr/ )

CRISPR-Cas Gene Editing Mechanism a. What is the meaning of CRISPR?

  • CRISPR is named as such because it contains clustered regularly interspaced short palindromic repeats, for which the name also stands for, and is a genomic feature by which the adaptive viral defense mechanism is dependent on (Klug et al. 2017).

b. How does CRISPR work in editing a gene?

  • CRISPR/Cas technology , adapted from the natural defense mechanisms of bacteria and archaea which use CRISPR-derived RNA and various CRISPR associated (cas) proteins, such as Cas9, to foil attacks by viruses or foreign bodies through chopping up and destroying these invader9s DNA; when transferred into other, more complex, organisms , allows for the manipulation of genes or <gene editing= (Vidyasagar 2018). Gene or Genome editing involves changing the DNA sequences in the genomes of various organisms so as to change the messages or instructions that are encoded within it (Vidyasagar 2018). This process can be done by inserting either a cut or break in the DNA, thus, tricking a cell9s natural DNA repair mechanisms into introducing the change that one wants. CRISPR-Cas9 then provides a means for it to be done so (Vidyasagar 2018).

increase yield when grown under drought stress (Chilcoat et al. 2017).

  • This application of CRISPR technology in the ARGOS8 gene of maize became very beneficial to the agricultural field, for it allowed the hybridized commercial maize to have a better performance and an increased yield even under drought stress compared to its wild type counterpart (Chilcoat et al. 2017).

Bibliography

Chilcoat D, Liu ZB, Sander J. 2017. Use of CRISPR/Cas9 for Crop Improvement in Maize and Soybean.Elsevier:doi/10.1016/bs.pmbts.2017.04.005.

Davis D. 2020. How do scientists <see= viruses? Part 2: qPCR. New York (NY): The Rockefeller University; [accessed 2020 Dec 20]. rockedu.rockefeller/component/qpcr/

Hist M. 2020. How B. scientists are addressing the COVID-19 testing challenge. Vancouver, BC: BC Cancer bcgsc; [updated 2020 May 22; accessed 2020 Dec 20]. bcgsc/news/how-bc-scientists-are-addressing-covid-19-testing-chall enge.

Jawerth N. 2020. How is the COVID-19 Virus Detected using Real Time RT-PCR? Vienna: International Atomic Energy Agency (IAEA); [updated 2020 March 27; accessed 2020 Dec 20]. iaea/newscenter/news/how-is-the-covid-19-virus-detected-usi ng-real-time-rt-pcr#:~:text=RT%E2%80%93PCR%20is%20a%20variation,RT %2C%20to%20allow%20for%20amplification.

Klug WS, Cummings MR, Spencer CA, Palladino MA. 2017. Essentials of

Genetics. 9th ed. Harlow: Pearson Education. pp. 492-495.

Kumar S, Nyodu R, Maurya VK, Saxena SK. 2020. Morphology,

Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Coronavirus Disease 2019 (COVID-19).doi/10.1007/978-981-15-4814-7_3.

Louten J. 2016. Features of Host Cells.

Elsevier:doi/10.1016/bs.pmbts.2017.04.005.

Neidler S. 2020. What are the differences between PCR, RT-PCR, qPCR,

and RT-qPCR? New York (NY): Enzo Life Sciences; [updated 2017 March; accessed 2020 Dec 20]. enzolifesciences/science-center/technotes/2017/march/what- are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr?/.

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SARS-Cov2 and Crispr-Cas Gene Assignment

Course: Genetics, Lecture (BIO130.01)

8 Documents
Students shared 8 documents in this course

University: Ateneo de Manila University

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Cracking the Code for SARS-Cov2 and the Discovery of CRISPR-Cas Gene
Editing Mechanism
SARS-Cov2
1.) Describe the morphological structure of SARS-Cov2 virus.
- According to studies through the use of Electron microscopy, the
structure of SARS-CoV-2 is speculated to be similar to that of
SARS-CoV with a virion size ranging from 70 to 90 nm (Kumar et al.
2020). The SARS-CoV-2 virus is also found to have surface viral
proteins, namely, the spike glycoprotein (S) which mediates interaction
with cell surface receptor ACE2, as well as, viral membrane (M) and
envelope (E) viral proteins that are embedded in host
membrane-derived lipid bilayer encapsulating the helical nucleocapsid
comprising viral RNA (Kumar et al. 2020).
2.) In a complete statement, state the size of the SARS-Cov2 genome.
- The size of the SARS-CoV-2 genome is found to be in the range of 26
up to 32 kb (kilobases) and is comprise of 6 to 11 open reading frames
(ORFs) encoding 9680 amino acid polyproteins (Kumar et al. 2020).
3.) How does the SARS-Cov2 infect (enter) its host cell and how is it
replicated inside the host cell ?
- The SARS-CoV-2 virus, like SARS-CoV, enters into its host target cells
through the binding of spike protein, which exhibits 10-20 times
higher affinity than that of SARS-CoV, to the
angiotensin-converting enzyme 2 (ACE2) receptor for
internalization and then the use of transmembrane serine proteases
(TMPRSS2) for S protein priming (Kumar et al. 2020). This then
results in conformational changes in the spike protein that leads to the
fusion of the viral envelope (E) protein with the host cell membrane
following the entry via an endosomal pathway (Kumar et al. 2020). This
internalization or entry is followed by the release of the viral RNA
into the host9s cytoplasm where it will undergo translation and
generate replicase polyproteins ppla and pplb which is then further
cleaved by the virus9 encoded proteinases into small proteins.
Replication of coronavirus which involves ribosomal frame-shifting
during the translation process then proceeds (Kumar et al. 2020).
Through discontinuous transcription, the replication process then
generates both genomic and multiple copies of subgenomic RNA
BIO 130.01 - MKP

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