Gene editing and Crispr Cas9 technology

2. CONTENT
 What is CRISPR-Cas9?
 Gene Editing
 Working of CRISPR-Cas9 Technology
 Application of CRISPR-Cas9 in Cancer Therapeutics
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3. WHAT IS CRISPR-Cas9? (KARIMIAN ET AL, 2019)
 Clustered Regularly Interspaced Short Palindromic
Repeats-CRISPR-associated nuclease 9 (CRISPR‐Cas9).
 The CRISPR-Cas9 system consist of single guide RNA (sgRNA) and DNA
endonuclease Cas9.
 CRISPR- Sections of genetic code containing short repetitions of base
sequences followed by spacer DNA segments.
 It was first identified in a prokaryotic defence system. 3
https://www.turbosquid.com/3d-models/crispr-cas9-3d-
1187179

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https://www.topperlearning.com/answer/why-restriction-enzymes-cut-dna-a-little-away-from-the-centre-of-pallindromic-
site/aala4099
https://medium.com/@ttlkhan/rewriting-ourselves-with-crispr-cdfae3dfd299
Spacer DNA

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 Cas9 (CRISPR associated protein 9) is a RNA- guided DNA
endonuclease enzyme associated with the CRISPR.
 Two distinct RNAs – the CRISPR targeting (crRNA) and the trans-
activating RNA (tracrRNA) – activate and guide Cas proteins to bind
DNA sequences which are subsequently cleaved.
 crRNAs are combined with tracrRNAs into a single guide RNA (sgRNA).
 It catalyzes site-specific cleavage of double stranded DNA.
 Cas9 has many applications in genetic engineering.

6. GENE EDITING
 Gene editing is a new technique, used to make specific and intentional
changes to DNA.
 Gene editing can be used to insert, remove and modify DNA in a
genome.
 All gene editing technologies involve an enzyme known as nuclease
for cutting the DNA.
 Its first use as a genome editing tool was performed in 2013 in
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7. WORKING OF CRISPR-Cas9 TECHNOLOGY
(KHALAF ET AL, 2020)
1) Complex of CRISPR-Cas9 enters the
nucleus of the cell.
2) Complex locks onto a short
sequence known as the PAM-
Protospacer adjacent motif.
3) Cas9 will unzip the DNA and match it
to its target RNA.
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https://gfycat.com/gifs/search/crispr-
cas9

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4) After the cut, cells try to repair the
cut.
• The repair may be Non-
homologues end joining (NHEJ) or
Homology directed repair (HDR).
• If repair takes place through NHEJ
mechanism than there is error-
prone repair- leads to mutation
that disables the gene.
• While in HDR mechanism, DNA
donor templates are used to
reconstruct cleaved DNA.
https://gfycat.com/gifs/tag/cas
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https://gfycat.com/gifs/tag/cas
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APPLICATION OF CRISPR-Cas9 IN CANCER THERAPEUTICS
 Cancer can be defined as a disease in which some of the body’s cells grow
uncontrollably and spread to other parts of the body.
 Cancer is second leading cause of death in the world.
 Treatments available for cancer are surgery, radiation and chemotherapy.
 Cancer is a genetic disease, gene therapy raises new hopes for cancer
therapeutics with minimum side effects with the use of gene editing
technology.
What is Cancer? (Mirza et al, 2019)
Application of CRISPR-Cas9 in Cancer Therapeutics

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CRISPR/Cas9 Genome Editing of Epidermal Growth Factor
Receptor (EGFR) Sufficiently Abolished Oncogenicity in
Anaplastic Thyroid Cancer. (Huang et al, 2018)
 Anaplastic carcinoma of the thyroid (ATC), also called undifferentiated
thyroid cancer, is the least common and most aggressive and deadly
thyroid gland malignancy of all thyroid cancers.
 Patients are usually in their 60s–70s at presentation, having an average
median survival of five months, and most patients with ATC do not live
one year from the day they are diagnosed.
CASE STUDY

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 ATC does not respond to radioactive iodine (131I) therapy, no effective
therapeutic options were available for patients with ATC resistant to
radioiodine.
 EGFR- Epidermal Growth Factor Receptor is a protein on cells that helps
to grow. Mutation in the gene of EGFR located on chromosome 7, makes
the cells grow too much that leads to cancer.
 In the present study, EGFR gene is being targeted because in anaplastic
thyroid cancer, it is found that EGFR is overexpressed in cancer cells, as
compared to normal thyroid cells.

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 Cell line- SW579 (Human gland epithelial squamous cell carcinoma cell
line)
EGFR Gene Targeting Using the CRISPR/Cas9 System
 Sequencing of Single-Guide RNA (sgRNA) Target Sites- Genomic DNA
was extracted, and PCR amplified the EGFR gene region.
 The vector used to transfer sgRNA was lentivirus and the technique
used to deliver vectors is Lentiviral transfection with CRISPR-Cas9
technique.
 Vector encodes for Cas9 protein and the specific sgRNA to EGFR DNA
Materials and Methods

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Two methods were used
for gene editing
Transduction with only
one sgRNA
No evidence of gene
editing was found
Transduction with two
sgRNAs- sgRNA_1 and
sgRNA_2
sgRNA_1 shows 88.2%
gene editing and sgRNA_2
shows 86.1% gene editing

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 The cells infected with second method resulted with decreased
levels of EGFR proteins.
 As a limitation of CRISPR-Cas9 technology, there is a need to
examine whether there is a off-target cleavage or not.
 Determination was carried out to check whether the CRISPR/Cas9
technology causes unexpected cleavage events at similar DNA
sequences.

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 No genomic editing was found with highly similar DNA sequence to
EGFR protein by sgRNA_1 and sgRNA_2.
 SW579 cell line was also evaluated by MTT assay to check the cell
growth with and without EGFR gene editing.
 Cell line with gene editing showed 40% reduced cell growth.
Result

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Application of CRISPR-Cas9 in Cancer Therapeutics
(Chen et al, 2019)
 Anticancer drug development
 Stromal-targeting therapies
 Cancer immunotherapy
 Oncolytic virotherapy

17. REFERENCE:
 Karimian A, Azizian K, Parsian H, Rafieian S, Shafiei‐Irannejad V, Kheyrollah M, Yousefi M, Majidinia M, Yousefi B.
CRISPR/Cas9 technology as a potent molecular tool for gene therapy. Journal of cellular physiology. 2019
Aug;234(8):12267-77.
 Khalaf K, Janowicz K, Dyszkiewicz-Konwińska M, Hutchings G, Dompe C, Moncrieff L, Jankowski M, Machnik M,
Oleksiewicz U, Kocherova I, Petitte J. CRISPR/Cas9 in cancer immunotherapy: animal models and human clinical trials.
Genes. 2020 Aug;11(8):921.
 Chen M, Mao A, Xu M, Weng Q, Mao J, Ji J. CRISPR-Cas9 for cancer therapy: Opportunities and challenges. Cancer
letters. 2019 Apr 10;447:48-55.
 Zhan T, Rindtorff N, Betge J, Ebert MP, Boutros M. CRISPR/Cas9 for cancer research and therapy. InSeminars in cancer
biology 2019 Apr 1 (Vol. 55, pp. 106-119). Academic Press.
 Mirza Z, Karim S. Advancements in CRISPR/Cas9 technology—Focusing on cancer therapeutics and beyond. In
Seminars in cell & developmental biology 2019 Dec 1 (Vol. 96, pp. 13-21). Academic Press.
 Huang LC, Tam KW, Liu WN, Lin CY, Hsu KW, Hsieh WS, Chi WM, Lee AW, Yang JM, Lin CL, Lee CH. CRISPR/Cas9
genome editing of epidermal growth factor receptor sufficiently abolished oncogenicity in anaplastic thyroid cancer.
Disease markers. 2018 Jan 1;2018.
 Chen M, Mao A, Xu M, Weng Q, Mao J, Ji J. CRISPR-Cas9 for cancer therapy: Opportunities and challenges. Cancer
letters. 2019 Apr 10;447:48-55.
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