Genetics Methods of CRISPR-Cas9 systems for treating sickle cell disease Essay

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For your final writing assignment of the semester, you are to compose a full research report. Keep in mind that everything we have done up to this point has been leading to this report. First, your annotated bibliography researched a topic / question of interest and provided you with the necessary background to speak on your topic. Then, your literature review should have identified trends in thinking, relations between your research, and gaps for further study that should form the basis and jumping point of your research. As for the research report itself, recall its basic elements as followed:

● Abstract (see “Article 9”)

● Introduction - partially includes your information from the literature review. - states your hypothesis and/or research questions.

● Methods (see “Article 7”) - details how you tested your hypothesis. -explains both your rationale for your methods and their limits.

● Results (see “Article 8”) - provides your raw (i.e., uninterpreted) data collected. – follows textual conventions and often “includes figures”.

● Discussion (see “Article 8”) - considers whether the data you obtained support the hypothesis. - acknowledge any data that was different from what you expected. - draws conclusions and supports any claim based upon your findings. -explore the theoretical and/or practical implications of your findings.

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This is what I did so far!!!!!! and I think I did not do well in it, I want you to change the thesis statement to This research aims to compare two different methods of CRISPR-Cas9 systems for treating sickle cell disease; switching the BCL11A gene off and Lentiglobin drug (Lovotibeglogen Autotemcel). Hypothesis is (( the BCL11A gene switching off is SUPERIOR in accuracy and sensitivity over the Lentiglobin drug (Lovotibeglogen Autotemcel)) Aysha YOU, can reword them in a better way than mine, please !!!!!!! Note: theYellow highlights at the end of each paragraph are the professor comments that highlight the mistake and how I can make it better. Please read the comments and try to fix those paragraphs based on what the professor said. The red highlight is just an indication that the yellow comment is belong to this parahraph. This is my literature review ↓↓↓↓↓↓↓↓↓the Lit review should be around 750 words ↓↓↓↓↓↓↓↓↓↓↓ Utilization of CRISPR-Cas9 in Treating Sickle Cell Diseases Sickle cell disease is one of the common inherited diseases that is identified by hemolytic anemia, pain, and swelling of the hands and feet. Nowadays, sickle cell disease (HbS) has several treatment options due to the advancement of gene therapy, such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated proteins), which functions as a gene-editing tool (Ribeil et al., 2017). CRISPR-Cas9 is classified into two classes, each of which is further subdivided into three subtypes. Additionally, each class of Cas9 can function optimally in several conditions. CRISPR-Cas9 can interact with the RNA to alter the strand properties for the gene organization. The CRISPR-Cas9 works as an effector module that can evolve as a defensive mechanism to target foreign RNA or DNA (Koonin & Makarova, 2019). This literature review aims to address the utilization of CRISPR-Cas9 systems in the treatment of sickle cell disease [The introduction is good. however this is too vague. This does not like literature review but more of an informative (summary-based document. It is unclear what your research question is even and overview of a topic is not even close to a research question/a literature review designed toward a research paper)]. CRISPR-Cas9 uses three different methods to treat sickle cell disease [It is hard to see how this could be a literature review. It seems like a purely informative document.]. The first method is by switching the BCL11A gene off. A mutation in the gene that expresses beta-globin gives rise to sickle cell anemia. This mutation will make the erythrocytes misshapen and become crescent-shaped cells. These sickle-shaped cells will aggregate and block blood vessels, which will result in low oxygen distribution to tissues (Yin et al., 2019). BCL11A is a gene that is highly expressed in humans, mostly in the hematopoietic cell systems and the brain. Hematopoietic cells are the stem cells that will differentiate into mature erythrocytes. (Esrick et al., 2021). This BCL11A gene is one of the switching genes in the human body. Switching genes means these genes can be switched on and off to be regulated. CRISPR-Cas9 is used to destroy the BCL11A enhancer, so the mutated gene will not be expressed and switched off (Yin et al., 2019). This BCL11A gene, when downregulated, will reduce sickle cells and their symptoms in patients. This downregulation will indirectly increase fetal hemoglobin (HbF) levels in the erythrocytes. Increasing fetal hemoglobin (HbF) levels is one of the successful therapeutic methods that have been used in treating sickle cell diseases. As a result, erythrocytes will express their regular shape and supply enough oxygen to the tissues (Yin et al., 2019). [Good information but it is just that- information. How do these sources relate to each other? Do they complement or contradict? Do they show a trajectory of research? How will you use them? How do they relate to your research question. This (and mostly only this) is what the literature review is about, not summary and certainly not an overview of some kind]. The second method that can be used to treat sickle cell disease is Lentiglobin (Lovotibeglogen Autotemcel), which is a type of CRISPR therapeutic drug. This is an important step to lessen or treat sickle cell disease because it can lead to vaso-occlusive and chronic hemolytic anemia (Pagliarulo, 2020). Using a modified in vivo method to extract a mutated beta-globin (HbB) stem cell and treat the mutated gene with Lentiglobin, then return the modified stem cell inside the body in the bone marrow (Kanter et al., 2022). Since the B-globin is modified, it will produce non-sickling hemoglobin that is called HbAT87Q (Pagliarulo, 2020). This way increases the production of healthy hemoglobin (HbA) that is synthesized in the body for the red blood cells component. As a result, red blood cells with the healthy hemoglobin HbAT87Q will have a normal biconcave shape, and the hemolysis of red blood cells will be reduced (Pagliarulo, 2020). This CRISPR Therapeutic with Lentiglobin is a one-time treatment that helps the patient regain the production of its healthy hemoglobin HbAT87Q instead of the mutated B-globin (HbB) that causes hemolysis of the red blood cells. [Great information and good grammar. However this is not a literature review. This reads like a research paper and not even an argumentative one at that. It is purely informative.] The third method for treating sickle cell disease can be induced by CRISPR-Cas9 technology through the elimination of the β-globin of sickle cell hemoglobin. According to Lin Ye that " the repairment of the genome via non-homology end joining (NHEJ) to generate an optimal genotype that could mimic fetal hemoglobin (HPFH), which will be producing a high-level of γglobin" (Ye et al., 2016). Eventually, the number of sickle hemoglobin decreased as a result. This method promotes the safest way to treat sickle cell disease via "autologous transplantation therapy" for the treatment of homozygous sickle cell disease (Ye et al., 2016) since most patients develop mild symptoms compared to the other methods [Great information and good grammar. However this is not a literature review. This reads like a research paper and not even an argumentative one at that. It is purely informative.] In conclusion, the use of CRISPR-Cas9 is still in an infancy stage with great potential that would shift the treatment procedure into a precise plan rather than a fixed method to ensure the optimal outcomes. In addition, the gene therapy plan for sickle disease can be through the inhibition of the BCL11A gene, the elimination of the theβ-globin of sickle cell hemoglobin or using the Lentiglobin drug to treat the mutated gene by in vivo method. Nevertheless, further investigation is required on CRISPR-Cas9 to reach the safest treatment that has fewer adverse reactions and is easy availability to those who need it [This has potential for a research review but it is just a Tack on at the end. You should focus the paper around something like this not mere summary]. References Esrick, E. B., Lehmann, L. E., Biffi, A., Achebe, M., Brendel, C., Ciuculescu, M. F., Daley, H., MacKinnon, B., Morris, E., Federico, A., Abriss, D., Boardman, K., Khelladi, R., Shaw, K., Negre, H., Negre, O., Nikiforow, S., Ritz, J., Pai, S.-Y., … Williams, D. A. (2021). Posttranscriptional genetic silencing ofbcl11ato treat sickle cell disease. New England Journal of Medicine, 384(3), 205–215. https://doi.org/10.1056/nejmoa2029392 Kanter, J., Walters, M. C., Krishnamurti, L., Mapara, M. Y., Kwiatkowski, J. L., Rifkin-Zenenberg, S., Aygun, B., Kasow, K. A., Pierciey, F. J., Bonner, M., Miller, A., Zhang, X., Lynch, J., Kim, D., Ribeil, J.-A., Asmal, M., Goyal, S., Thompson, A. A., & Tisdale, J. F. (2022). Biologic and clinical efficacy of lentiglobin for sickle cell disease. New England Journal of Medicine, 386(7), 617–628. https://doi.org/10.1056/nejmoa2117175 Koonin, E. V., & Makarova, K. S. (2019). Origins and evolution of CRISPR-Cas systems. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772), 20180087. Pagliarulo, N. (2020, June 12). NEW CRISPR, gene therapy results strengthen potential for treatment of blood diseases. BioPharma Dive. Retrieved March 17, 2022, from https://www.biopharmadive.com/news/crispr-vertex-bluebird-sickle-cell-thalassemia-studyupdate/579692/ Ribeil, J.-A., Hacein-Bey-Abina, S., Payen, E., Magnani, A., Semeraro, M., Magrin, E., Caccavelli, L., Neven, B., Bourget, P., El Nemer, W., Bartolucci, P., Weber, L., Puy, H., Meritet, J.-F., Grevent, D., Beuzard, Y., Chrétien, S., Lefebvre, T., Ross, R. W., … Cavazzana, M. (2017). Gene therapy in a patient with sickle cell disease. New England Journal of Medicine, 376(9), 848–855. https://doi.org/10.1056/nejmoa1609677 Ye, L., Wang, J., Tan, Y., Beyer, A. I., Xie, F., Muench, M. O., & Kan, Y. W. (2016). Genome editing using CRISPR-Cas9 to create the HPFH genotype in hspcs: An approach for treating sickle cell disease and β-thalassemia. Proceedings of the National Academy of Sciences, 113(38), 10661–10665. https://doi.org/10.1073/pnas.1612075113 Yin, J., Xie, X., Ye, Y., Wang, L., & Che, F. (2019). BCL11A: A potential diagnostic biomarker and therapeutic target in human diseases. Bioscience Reports, 39(11). https://doi.org/10.1042/bsr20190604 ↓↓These below are extra references if you want to use any of them!!! If you want also you can use other ones that you may find!! U are not limited to this. ↓↓ Fogleman, S., Santana, C., Bishop, C., Miller, A., & Capco, D. G. (2016, August 20). CRISPR/Cas9 and mitochondrial gene replacement therapy: Promising techniques and ethical considerations. American journal of stem cells. Retrieved February 18, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043096/#b40 (Fogleman et al., 2016) Kang, X. J., Caparas, C. I., Soh, B. S., & Fan, Y. (2017). Addressing challenges in the clinical applications associated with CRISPR/Cas9 technology and ethical questions to prevent its misuse. Protein & Cell, 8(11), 791–795. https://doi.org/10.1007/s13238-017-0477-4 (Kang et al., 2017) Esrick, E. B., Lehmann, L. E., Alessandra, B., Achebe, M., Brendel, C., Ciuculescu, M. F., . . . Manis, J. P. (2021). Post-transcriptional genetic silencing of BCL11A to treat sickle cell disease. The New England Journal of Medicine, 384(3), 205-215. doi:http://dx.doi.org/10.1056/NEJMoa2029392 (Esrick et al., 2021) Song X, Liu C, Wang N, Huang H, He S, Gong C, Wei Y. Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy. Adv Drug Deliv Rev. 2021 Jan;168:158-180. doi: 10.1016/j.addr.2020.04.010. Epub 2020 May 1. PMID: 32360576. Koonin, E. V., & Makarova, K. S. (2019). Origins and evolution of CRISPR-Cas systems. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 374(1772), 20180087. https://doi.org/10.1098/rstb.2018.0087 (Koonin & Makarova, 2019) Baddeley, H. J., & Isalan, M. (2021). The application of CRISPR/CAS systems for antiviral therapy. Frontiers in Genome Editing, 3. https://doi.org/10.3389/fgeed.2021.745559 (Baddeley & Isalan, 2021) Wilson, L. O. W., O’Brien, A. R., & Bauer, D. C. (2018). The Current State and Future of CRISPR-Cas9 gRNA Design Tools. Frontiers in Pharmacology, 9. https://doi.org/10.3389/fphar.2018.00749 Stadtmauer, E. A., Fraietta, J. A., Davis, M. M., Cohen, A. D., Weber, K. L., Lancaster, E., Mangan, P. A., Kulikovskaya, I., Gupta, M., Chen, F., Tian, L., Gonzalez, V. E., Xu, J., Jung, I.young, Melenhorst, J. J., Plesa, G., Shea, J., Matlawski, T., Cervini, A., … June, C. H. (2020). CRISPR-engineered T cells in patients with refractory cancer. Science, 367(6481). https://doi.org/10.1126/science.aba7365 Song, X., Liu, C., Wang, N., Huang, H., He, S., Gong, C., & Wei, Y. (2021). Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy. Advanced drug delivery reviews, 168, 158–180. https://doi.org/10.1016/j.addr.2020.04.010 Creating Research Questions Broad Research Topic The subject area to be investigated Research Problem The issue or problem within the broad topic area (gap) Once you have gathered enough knowledge on the topic you want to pursue, you can start focusing on a more specific area of study. One option is to focus on gaps in existing knowledge or recent literature. Research Purpose The statement of the intent or the objective of the study What gap(s) are you going to investigate specifically. Research Question Question(s) about a process, issue or phenomenon to be explored Specific question(s) on the particular gap you are trying to investigate. Hypothesis A Hypothesis is an assumption or prediction of your research question (S). This is what you are trying to investigate in order to see if it is true or false. Either way, we are seeking for the truth. INSTRUCTIONS !!!!!!!!!!!!!!!!!!!!!!!!!! Research Report For your final writing assignment of the semester, you are to compose a full research report. Keep in mind that everything we have done up to this point has been leading to this report. First, your annotated bibliography researched a topic / question of interest and provided you with the necessary background to speak on your topic. Then, your literature review should have identified trends in thinking, relations between your research, and gaps for further study that should form the basis and jumping point of your research. As for the research report itself, recall its basic elements as followed: ● Abstract (see “Article 9”) ● Introduction - partially includes your information from the literature review. - states your hypothesis and/or research questions. ● Methods (see “Article 7”) - details how you tested your hypothesis. -explains both your rationale for your methods and their limits. ● Results (see “Article 8”) - provides your raw (i.e., uninterpreted) data collected. – follows textual conventions and often “includes figures”. ● Discussion (see “Article 8”) - considers whether the data you obtained support the hypothesis. - acknowledge any data that was different from what you expected. - draws conclusions and supports any claim based upon your findings. -explore the theoretical and/or practical implications of your findings. U CAN USE GRAPHES !!!!!!!! You will be graded on two primary axes: first, your ability to follow the advice and conventions for each component as identified in our course-pack; and second, your ability to articulate your argument, processes, and logical progressions throughout the assignment. Assignment Details: -You should aim for a length of roughly 1000 words (try not to go too far beyond 1300 words). the subsequent presentation
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Methods of CRISPR-Cas9 systems for treating sickle cell disease

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Abstract
Sickle Cell Anemia (SCA) was mentioned in literary works for the first time over a
century ago. The discovery of its genetic foundation triggered a flurry of genetic and
biochemical investigations that aided in the comprehension of its pathogenesis. Regrettably,
translating this information into therapeutic development has been excessively sluggish and
difficult. In the last decade, the exploration of BCL11A, a major -globin gene suppressor, has
resulted in a greater knowledge of the transition from fetal to adult hemoglobin and a reemergence of interest in therapeutical and biological strategies for reactivating fetal hemoglobin
as promising clinical alternatives. Along with medicinal stimulation of fetal hemoglobin,
advances in cell therapy, mixed chimerism, genetic manipulation, and genomics have been very
optimistic results. Other advancements have resulted in the Regulatory approval of three new
pharmaceuticals for the therapy of sickle cell anemia between 2017 and 2019, with numerous
more now under investigation. This research compares two methods of CRISPR-Cas9 systems
for treating sickle cell disease; switching the BCL11A gene off and Lentiglobin drug
(Lovotibeglogen Autotemcel).
Keywords: “BCL11A," " Lentiglobin for sickle cell disease," “Origins and evolution of
CRISPR-Cas systems,” “sickle cell disease”

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Introduction
SCA is the most prevalent monogenic disorder globally, with around 60,000 cases of
TDT and 300,000 cases of SCD diagnosed each year. Both differences are characterized by gene
defects encoding the hemoglobin component (HBB). TDT-causing mutation in Gene mutation
results in decreased (+) or absent (0) -globin production and a mismatch here between -like and like beta globin of hemoglobin, resulting in inefficient erythrocytes. Missense mutations cause
sickle anemia in HBB that substitutes valine for glutamic acid at amino acid residue. Gelation of
deoxygenated unstable hemoglobin results in erythrocyte distortion, hemolytic anemia, and
thrombocytopenia, as well as severe vaso-occlusive events, permanent end-organ destruction, as
well as and early death.
Researchers have documented single-nucleotide polymorphisms (SNPs) related to higher
fetal hemoglobin production in adulthood. Several of these SNPs are situated on the human
chromosome at the BCL11A gene and are related to a decreased incidence of TDT and SCD.
BCL11A is a zinc finger–containing signaling pathway that inhibits the synthesis of -globin as
well as fetal hemoglobin in hematopoietic cells; these SNPs linked with hemolytic anemia are
positioned in an erythroid-specific promoter to inhibit BCL11A manifestation while increasing
fetal hemoglobin manifestation.
The CRISPR-Cas9 nuclease response, a microbial response adept at cleaving
bacteriophage or plasmids, effectively targets point mutations (indels) to specified DNA
Fragment sites. To mimic the genetic durability of fetal hemoglobin, we employed CRISPRCas9 genome engineering methods in hematopoietic st...


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