CSPR-CAS9 SYSTEM

Genome engineering using the CRISPR-Cas9 system. Sarah Ahmed Elsharawy - 195611 Rahaf Fareed Mansour - 195731 Nashwa Sherif Zarif - 192911 Alaa Nasser Salaheldin - 192243 Doaa Abdelrazek - 192145 Mariem Emadeldin - 194399 Nada Fouad Hosny - 194471.

Name ID Task Sarah Ahmed Elsharawy 195611 Discovery of system & Powerpoint Formatting Rahaf Fareed Mansour 195731 Principle of the system Nashwa Sherif Zarif 192911 Principle of the system Alaa Nasser Salaheldin 192243 Applications of the system Doaa Abdlrazek 192145 Applications of the system Mariem Emadeldin 194399 Future Perspectives Nada Fouad Hosny 194471 Future Perspectives.

Table of Contents. Discovery. Principle. Applications.

Discovery of CRISPR-Cas9 System (Gostimskaya , 2022).

Discovery of CRISPR-Cas9 System cont. (Gostimskaya , 2022).

Discovery of CRISPR-Cas9 System cont. (Gostimskaya , 2022).

Discovery of CRISPR-Cas9 System cont. (Gostimskaya , 2022).

Discovery of CRISPR-Cas9 System cont. (Gostimskaya , 2022).

Principle of CRISPR-Cas9 System. CRISPR stands for (clustered regularly interspaced short palindromic repeat).​.

Matching genomic sequence DNA DELETING A GENE Gene is disrupted Guide RNA Casg INSERT A GENE Gene has a new sequence.

Detection of Low-frequency mutant DNA, samples with long and short fragment deletion and the single nucleotide polymorphisms(SNP). ​ Most mutations related to diseases are deletion mutation, SNP and DNA mutations are related to the development, diagnosis, progression and therapy of diseases.​.

Application for CRISPR/Cas9 system cont.:. CRISPR/Cas9 system has the ability to treat many genetic and viral diseases like:​ Cystic Fibrosis; For treatment, intestinal stem cell is isolated and develop it in culture in laboratory and then remove the target allele (F508) and placed back to the human .​ Huntington; in this disease treatment, CAG repetitions present in HTT gene can be removed by CRISPR/CAS9.​ Rett Syndrome and Schizophrenia ​.

Application for CRISPR/Cas9 system cont.:. The Cas9 protein and sgRNA can be also used for multiplex gene editing on the relevant target, which will realize the full utilization of the experimental materials, also used in gene expression regulation and gene functional studies in diverse species; in zebra fish, mice, drosophila and bacteria .​ The most widely used Streptococcus pyogenes CRISPR/Cas9 system identifies the homologous target by recognition of a trinucleotide “NGG” protospacer adjacent motif (PAM) sequence, which appears about every 8 bases in the genome of human. CRISPR/Cas9 shows precise in vitro DNA cleavage. ​.

Application for CRISPR/Cas9 system cont. :. ​ CRISPR/Cas system has also been used in biotechnology and pharmacology. It plays important role in making vaccines for Pseudorabies virus.​ CRISPR-Cas9 can able to treat HIV patients. Hepatitis B is also treated by CRISPR-Cas9 system. In this disease, covalently closed circular DNA (cccDNA) present that produces a new type of antigen and it can cause hepatitis B.

Application for CRISPR/Cas9 system cont.:. This diverse system is also used in plants for many purposes like gene mutation, gene knock-in, gene knock-out, epigenomes and modifications.​ In Arabidopsis, several genes (AtPDS3, AtFLS2, AtADH, AtFT, AtSPL4, AtBRI1) are targeted by using mutational efficiencies and these mutations are transferred to their offspring successful..

Application for CRISPR/Cas9 system cont. :. Researchers produce catalytically inactive Cas9 protein; name dead Case or dCas9, by point mutation in HNH and RuvC domains of Cas9 enzyme.​ dCas9 forms a complex with CRISPRi and disturbs the functional sites of transcription that controls the transcription process. Then this complex interferes in the elongation steps of transcription and express or repress the target sequence..

Future Perspectives cont.. Sickle cell disease in an inherited monogenic disorder that can be fatal. ​ There are currently only two FDA approved medication that will only lessen the severity of the disease. ​ Due to the programmable nucleases, the SCD is a potential candidate for CRISPER Cas9 gene editing.​ CRISPER Cas9 is able to correct the mutation of SCD or increase the fetal haemoglobin expression.​ These is not feasible now due the off-target effect and subtherapeutic effect.(Demirci et al., 2019)​.

Future Perspectives cont.. Genetic engineering using CRISPR-Cas9 ​.

Future Perspectives cont.. Role of CRISPER-Cas9 chemotherapy.

In plants, The CRIPSR/Cas9 system is currently being developed and applied toward engineering traits of fruit crops: The rapid development of CRISPR/Cas9-based tools may help overcome some of the barriers in horticultural crops. Methods that reduce the need for multi-generation cycles or that increase editing efficiency are highly desirable. Fast and low-cost mutant screening is an area of urgent need. Targeting cis-elements to alter gene regulation is a clever way to generate allelic series as demonstrated in citrus LOB1 gene and tomato CLV3 gene..

Strategies that aid in the production of transgene-free edited fruit crops will be vital to consumer acceptance and commercialization The importance of public dialog on genome editing and investment on consumer-friendly traits such as healthier products and longer shelf-life. The researchers will be interest for the wild type plants and preserve the extended plants through the microspore. Boost the future desired mutation of the plants for modify the eukaryotic and prokaryotic genome..

References. Razzaq, A., & A. Masood. (2018) "CRISPR/Cas9 system: a breakthrough in genome editing." Mol Biol .7 (2). doi: 10.4172/2168-9547.1000210.‏​​ Jia, C., Huai, C., Ding, J., Hu, L., Su, B., Chen, Hongyan &Lu, D.(2018). New applications of CRISPR/Cas9 system on mutant DNA detection. Gene. 641, 55-62. doi:10.1016/j.gene.2017.10.023​ Chang N, Sun C, Gao L, Zhu D, Xu X, et al. (2013) Genome editing with RNAguided Cas9 nuclease in Zebrafish embryos. Nat Publ Gr 23: 465-472. Demirci, S., Leonard, A., Haro-Mora, J.J., Uchida, N. and Tisdale, J.F. (2019). CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges. Advances in Experimental Medicine and Biology , 5. doi:10.1007/5584_2018_331.​ Manghwar, L. Xianlong, Z. Shuangxia E, panel et al. (2019) CRISPR/Cas System: Recent advances and future prospects for genome editing, Trends in Plant Science. Elsevier Current Trends. Available at: https://reader.elsevier.com/reader/sd/pii/S1360138519302432?token=4F7A468CC769CA03A6ED47F820D2BF5939E5CEEFB2DF25649870DBE79C55A084F971F07FA835CA9AB5634749ADF07E05&originRegion=eu-west-1&originCreation=20221102230142 (Accessed: November 3, 2022). ​ Tiruneh G/Medhin, M. et al. (2021) Current applications and future perspectives of CRISPR-Cas9 for the treatment of lung cancer, Biologics : targets & therapy. Dove. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178582/ (Accessed: November 3, 2022).​ Gostimskaya, I. (2022) “CRISPR–cas9: A history of its discovery and ethical considerations of its use in genome editing,” Biochemistry (Moscow), 87(8), pp. 777–788. Available at: https://doi.org/10.1134/s0006297922080090..