This presentation is done by Group 8, for the module Gene and Tissue Culture Technology. In Taylors University Lakeside Campus, Selangor, Malaysia.

1. ALAA KHAMIS (0325098)
SARAH LUCAS (0328873)
MARYAM HAKI ( 0327530)
AZLYNA BINTI MOHD NOOR
(0322163)
PAK WEN (0327357)
U
Applications of Gene
editing: CRISPR-Cas9 in
Cancer Therapy
(Oncogenes)

2. Most of the diseases occurring in this day and time are caused by
abnormal sequence or mismatches sequences in our genomic structure.
Gene editing is a technique used to as the new model to cure diseases by
changing an organism's DNA in either expressing or suppressing a gene,
or totally replacing it. This can lead to avoid genetic diseases,
transmitted diseases or diseases arising from mutated sequences.
CRISPR is the new gene editing technology used to modify genetic
sequences to cure genetic mutations or mismatched sequences. It
stands for Clustered Regularly Interspaced Short Palindromic Repeats,
it arises from the bacterial defense systems against viral infections
(Zhang, 2013) CRISPR-Cas9 is a natural DNA-sniping enzyme in
bacteria, where Cas9 is (CRISPR-associated protein 9). It works by
targeting a specific stretch of the genetic code known as a spacer that is
transcribed into a short RNA sequence (crRNA) which guides the system
to match the sequence and edit the DNA precisely at specific locations
leading to the modification of the genes in the living cells and organisms
(Ball, 2016)
EASTWAY UNIVERSITY
OF SOCIAL SCIENCES
WHAT DO YOU NEED TO KNOW ABOUT CRISPR-CAS9?
Source:https://www.cambridge.org/core/journals/mrs-
bulletin/news/crispr-implications-for-materials-science

3. Cancer is a common disease that develops from cumulative genetic and
epigenetic alterations, that are induced from environmental factors, or
random mutations at cellular level.
In cancer treatment it has been a challenge to identify the driver
mutations and oncogenes to develop a therapeutic effect, however viral
driven cancer cells are highly expressive to viral oncogenes which are
genes that can cause mutations, the oncogene dependent cancer cells
express a single or multiple gene to enhance survival and growth, this
notion was discovered by Bernard Weinstein in 2000, after observing
how multiple epigenetic and genetic abnormalities do not interfere in
the process of cancer development, because only one single gene is
responsible for activation or inactivation of the cell, and can be
inactivated to halt the cancer growth and survival (Jubair et. McMillan,
2017).
This serves as a model for CRISPR-Cas9 in cancer therapeutic
approaches, where the model allows testing of both preclinical and
clinical conditions to examine the efficiency of gene editing. CRISPR-
Cas9 has been used in modelling oncogenic mutations in cell lines, adult
animals, and combat cancer. It works by either disabling the oncogenic
viruses or by cancer genome manipulation.
IS CRISPR-CAS9 THE SOLUTION TO A CANCER FREE GENERATION?
Source:http://scienceblog.cancerresearchuk.o
rg/2016/02/01/crispr-gene-editing-new-
chapter-in-cancer-research-or-blot-in-the-
ethical-copybook/

4. Objectives
1 2 3 4
To study the
performance,
advantages and
disadvantages
of Gene
engineering
technology in
the field of
cancer therapy.
To study the
principles and
applications of
CRISPR-Cas 9
in the field of
Cancer
therapy.
To understand
the mechanism
applied in
CRISPR-Cas9 in
the treatment of
Oncogene
addicted cancer
cells.
To research on
the current
development
and the future
developments
of CRISPR-Cas9
in the field of
Cancer therapy
and treatment
potential.

5. How does CRISPR-Cas9 work ?
The CRISPR-Cas9 system consists of two key molecules that introduce a change
(mutation )into the DNA. These are:
1- ENDONUCLEASE enzyme called Cas9, responsible for the double-stranded
breaks at the site, when targeted by a guide RNA and Cleavage occurs on both
strands (Yourgenome 2016).
2- a piece of RNA called guide RNA (gRNA),makes sure that the Cas9 enzyme
cuts at the right point in the genome (Yourgenome 2016).
Fig. 3: CRISPR-Cas9 targeting system. In the CRISPR/Cas9 system, a guide
RNA hybridizes a 20-nt DNA sequence immediately preceding an NGG DNA
motif (protospacer-associated motif or PAM), resulting in a double-strand
break (DSB) 3 bp upstream of the NGG. The double-stranded DNA breaks
become substrates for endogenous cellular DNA repair machinery that
catalyze nonhomologous end joining (NHEJ) or homology-directed repair
(HDR) ((Zhuchi et al. 2015).
source: https://www.researchgate.net/figure/CRISPR-Cas9-
targeting-system-In-the-CRISPR-Cas9-system-a-guide-RNA-
hybridizes-a-20-nt_fig1_280694279?
_sg=AX_6k8nwScCS87yRLqMWNZl3EEH-
E02NUlGxjkVNtrD0vt8BsQWHbLBQDQLjChwVdohQ0PHsoxHh
unV71ynYfw

6. Fig.4: In vivo and in vitro gene editing
therapeutic strategies for CRISPR-
Cas9-based cancer therapy. A. In in
vivo gene editing therapy,
Cas9/sgRNA complex are systemic or
targeted delivered into the patient
via either viral or nonviral vehicles
for gene editing in cancer therapy, for
example, (epi)genome modification or
transcriptional regulation of cancer
cells. B. In in vitro gene editing based
immunotherapy, patient-derived
immune cells are isolated and genetic
modified with CRISPR-Cas9 system,
and then infused back into the same
patient; for oncolytic therapy,
oncolytic viruses are edited and
introduced into patients to kill cancer
cells (Yi et al. 2016)
HOW IS IT DELIVERED TO PATIENTS?
source: https://www.sciencedirect.com/s
cience/article/pii/S0304419X16300634

7. Figure 4: In vivo effects of CRISPR-mediated
editing of the BCR/ABL oncogene. (A) Tumor
growth (mm3) over the 24 days following
subcutaneous cell injection. Similar tumor
growth was observed in Boff-p210 (grey bars)
and Boff-p210 Cas9 mock sgRNA (black dots
line) injected cells. Tumors formed by Bcr-Abl-
EP cells (black squares line) were half the size
of those induced previously. Tumor growth was
observed when the single edited cell-derived
cells were injected (grey dotted line), as well
Baf/3 cells (dark grey bars). The plot shows
medians and ranges; ***p < 0.001). (B) After 24
days, mice were sacrificed and their tumor
mass measured. The final tumor mass was
reduced by half in the case of the edited pool
cells (black squares), relative to controls (black
dots). Bcr-Abl-SC (grey dots) and Baf/3 (black
triangles) cells were unable to form a
subcutaneous tumor. The plot shows medians
and ranges; **p < 0.05). (C) External appearance
of mice and developed tumors 24 days after
subcutaneous cell injection.
(García-Tuñón et al., 2017)
Recent studies on oncogene & the
effects of CRISPR/CAS9 on
chronic myeloid leukemia
CML in a xenograft model
source: http://www.oncotarget.com/index.php?
journal=oncotarget&page=article&op=view&path%5
B0%5D=15215&path%5B1%5D=48659#R29

8. Applications of CRISPR-Cas9
1. CRISPR/Cas9 mediated gene knockout - production of knockout (KO)
cell lines by disrupting one or more oncogenes or tumor suppressors
concurrently (Yue et. al, 2016). Carried out using lentiviral vector shown in
Figure 5.
Example:
a-Prostate cancer cells, DU145, has its malignant potential significantly
decrease when Nanog and Nanogp8 are knocked out (Kawamura et al.,
2015).
b- Lung tumor growth in Cas9 transgenic mice significantly increases when
the Kras oncogene, p53 and Lkb1 are knocked out while a point mutation in
Kras G12D at the genomic locus is inserted (Platt et al., 2014).
Figure 5: Lentiviral vector based
CRISPR/Cas9 transcriptional activation or
repression (Yue et. al, 2016)

9. Applications of CRISPR-Cas9
2. CRISPR/Cad9 mediated transcriptional regulation - usage of CRISPR-Cas9 to mediate transcriptional
activation and repression (Yue et. al, 2016).
Transcription activation and inhibition libraries are available as a result of this for cancer gene functional studies.
Example:
a- CRISPRa and CRISPRi human genome wide libraries created to identify genes essential for gene survival and
activation (Gilbert et al., 2014)
3- CRISPR/Cas9 mediated chromosome translocation - can be used to simulate chromosome translocation in a
number of human cancers.
Example:
a- pax3-foxo1 fusion gene in human alveolar rhabdomyosarcoma (Lagutina et al., 2015)
b- BCAM-AKT2 fusion gene in ovarian serous carcinoma (Kannan et al., 2015)
c- EML4-ALK fusion gene in lung cancer (Maddalo et al., 2014)

10. Advantages and Disadvantages of CRISPR-Cas9
Target design simplicity - Relies on ribonucleotide
formation and not protein/DNA formation
(Yeadon, 2018).
Efficiency - Directly injecting RNAs encoding the
CAS protein and gRNA into mouse developing
mouse embryos (Yeadon, 2018).
Multiplexed Mutations - Multiple genes at the
same time by injecting them with multiple gRNAs
(Yeadon, 2018).
CRISPR is capable of modifying chromosomal
targets with high fidelity (Synbio Technologies,
n.d.)
Cost-effective and easy-to-use technology (Synbio
Technologies, n.d).
Off-site effects - Difficult to identify and require
scanning the genome for mutations at site with
sequence similarity to the gRNA target sequence
(Yeadon, 2018).
Mosaicism - Mice with a mutant allele in only some
of their cells can be produced (Yeadon, 2018).
Multiple alleles - Healing of the nuclease cleavage
site by non-homologous end joining can be
produced cohorts of mice with different mutation
from the same targeting constructs, requires
genome sequencing in order to verify the nature of
the specific mutation (Yeadon, 2018).
CRISPR can only recognize genetic sequences to
around 20 bases long (Woollaston, 2018).
Advantages Disadvantages

11. Future Development:
Human papillomaviruses (HPVs) are the causative
agents of almost all cervical carcinomas.
The HPV DNA genome integrates into the cellular genome, where it expresses high levels of
two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability.
Bacterial CRISPR/Cas RNA-guided endonuclease was found to have the potential to be
reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed
by HPV, resulting in cell cycle arrest and eventual death of the cancer cells.
Researchers therefore proposed that viral vectors designed to deliver E6- and/or E7-specific
CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and
eliminate HPV-induced cancers.

12. Future Development:
As many as a fifth of all human cancers are caused
by viruses which encodes viral oncogenes that
promote carcinogenesis.
Oncogenes drive cell proliferation by a gain-of-
function ability to stimulate cell signaling pathways
inappropriately.
CRISPR Cas9 can be used in the inactivation of an oncogene by the disruption of a protein motif that is
necessary for the activity of the oncoprotein.
For example, the src family of oncogenes requires tyrosine kinase activity to transform, hence it could be
targeted by CRISPR/Cas9 directed towards the tyrosine kinase domain.
Some examples of viruses that expresses oncogenes associated with human cancer that can be a potential
target for CRISPR/Cas9 includes: Hepatitis B virus (HBV) and Epstein-Barr virus (EBV).

13. Future Development:
Cellular genes that are mutated to become oncogenes also have a huge potential as targets for treating human
cancer.
Oncogene changes occur in many cancers and are an important driving force for malignant cell proliferation.
CRISPR/Cas9 could be targeted against the mutated form of the cellular oncogene to disrupt and inactivate it.
For example, non-receptor tyrosine kinase can be inappropriately activated by mutations in their regulatory
domains. These oncogenes could be targeted using CRISPR/Cas9 and gRNA directed against the tyrosine kinase
domain, which is necessary for oncogenic activity, and inactivates it to stop the cancerous activity.
The CRISPR/Cas9 system has the potential to be developed even further to provide a specific and efficacious
approach against many types of oncogenic changes in cancer cells.

14. Future Development:
The study explored the ability of CRISPR/Cas9 technology to obliterate BCR-ABL fusion in order to determine its
impact on the leukemic processes in in vitro and in xenograft models of Chronic Myeloid Leukemia (CML).
Results found that CRISPR/Cas9 genomic editing technology managed to allow the ablation of the BCR/ABL fusion
gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo
xenograft model of CML.
CRISPR/Cas9 technology can therefore be used as a new therapeutic tool that overcomes resistance to the usual
treatments for CML patients in the future.

15. Conclusion:
CRISPR Cas9 can be used in the inactivation of an oncogene by the disruption of a protein motif
that is necessary for the activity of the oncoprotein.
The CRISPR/Cas9 system has the potential to be developed even further to provide a specific and
efficacious approach against many types of oncogenic changes in cancer cells.
CRISPR/Cas9 technology can therefore be used as a new therapeutic tool that overcomes
resistance to the usual treatments for CML patients in the future.

16. References:
Feng Zhang, 2017. ‘Questions and Answers about CRISPR’ Broad Institute. Accessed 14th of May 2018. Available at: https://www.broadinstitute.org/what-broad/areas-
focus/project-spotlight/questions-and-answers-about-crispr
García-Tuñón, I., Hernández-Sánchez, M., Ordoñez, J., Alonso-Pérez, V., Álamo-Quijada, M., Benito, R., Guerrero, C., Hernández-Rivas, J. and Sánchez-Martín, M. 2017, The
CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia, oncotraget, viewed 19 May 2018,
<http://www.oncotarget.com/index.php?journal=oncotarget&page=article&op=view&path%5B0%5D=15215&path%5B1%5D=48659>
Gilbert, L., Horlbeck, M., Adamson, B., Villalta, J., Chen, Y., Whitehead, E., Guimaraes, C., Panning, B., Ploegh, H., Bassik, M., Qi, L., Kampmann, M. and Weissman, J., 2014,
‘Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation’, Cell, vol. 159 no. 3, pp.647-661, viewed 19 May 2018,
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253859/>
Kannan, K., Coarfa, C., Chao, P., Luo, L., Wang, Y., Brinegar, A., Hawkins, S., Milosavljevic, A., Matzuk, M. and Yen, L. 2015, ‘Recurrent BCAM-AKT2fusion gene leads to a
constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma’, Proceedings of the National Academy of Sciences, vol. 112 no. 11, viewed 20 May 2018,
<https://www.ncbi.nlm.nih.gov/pubmed/25733895>
Kawamura, N., Nimura, K., Nagano, H., Yamaguchi, S., Nonomura, N. and Kaneda, Y. 2015, ‘CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the
malignant potential of prostate cancer cells’, Oncotarget, vol. 6, no. 26, viewed 19 May 2018, <http://www.oncotarget.com/index.php?
journal=oncotarget&page=article&op=view&path[]=4293&path[]=10990>
Lagutina, I., Valentine, V., Picchione, F., Harwood, F., Valentine, M., Villarejo-Balcells, B., Carvajal, J. and Grosveld, G. 2015, ‘Modeling of the Human Alveolar
Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nucleas’, PLOS Genetics, vol. 11 no. 2, viewed 20 May 2018,
<https://www.ncbi.nlm.nih.gov/pubmed/25659124>
Kennedy, M.K et al. 2014, Inactivation of the Human Papillomavirus E6 or E7 Gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RHNA-Guided Endonuclease,
Journal of Virology, vol. 88, no. 20, pp. 11965-11972, viewed on 21 May 2018, <http://jvi.asm.org/content/88/20/11965.short>

17. References:
Lagutina, I., Valentine, V., Picchione, F., Harwood, F., Valentine, M., Villarejo-Balcells, B., Carvajal, J. and Grosveld, G. 2015, ‘Modeling of the Human Alveolar
Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nucleas’, PLOS Genetics, vol. 11 no. 2, viewed 20 May 2018,
<https://www.ncbi.nlm.nih.gov/pubmed/25659124>
Maddalo, D., Manchado, E., Concepcion, C., Bonetti, C., Vidigal, J., Han, Y., Ogrodowski, P., Crippa, A., Rekhtman, N., de Stanchina, E., Lowe, S. and Ventura, A., 2014, ‘In vivo
engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system’, Nature, vol. 516 no. 7531, viewed 20 May 2018,
<https://www.ncbi.nlm.nih.gov/pubmed/25337876>
Platt, R., Chen, S., Zhou, Y., Yim, M., Swiech, L., Kempton, H., Dahlman, J., Parnas, O., Eisenhaure, T., Jovanovic, M., Graham, D., Jhunjhunwala, S., Heidenreich, M., Xavier, R.,
Langer, R., Anderson, D., Hacohen, N., Regev, A., Feng, G., Sharp, P. and Zhang, F., 2014, ‘CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling’, Cell, vol. 159 no.
2, pp.440-455, viewed 19 May 2018, <https://www.ncbi.nlm.nih.gov/pubmed/25263330>
Philip Ball, 2016. ‘CRISPR: Implications for materials science’, The Ascent of CRISPR. Biological biomaterials, Cambridge Core. Accessed 12th of May 2018. Available at
https://www.cambridge.org/core/journals/mrs-bulletin/news/crispr-implications-for-materials-science
Synbio Technologies, n.d., What Are The Advantages Of CRISPR-Cas9?, viewed 19th March 2018 <http://www.synbio-tech.com/crispr-cas9-advantages/>
Tunόn, I.G et al, 2017, The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia,
Oncotarget, vol. 8, pp. 26027-26040 , viewed on 22 May 2018, <http://www.oncotarget.com/index.php?
journal=oncotarget&page=article&op=view&path%5B0%5D=15215&path%5B1%5D=48659>
Woollaston, V, 2018, The Great Barrier Reef could be saved from climate change thanks to controversial gene-editing tool CRISPR, viewed at 20th March 2018
<http://www.alphr.com/bioscience/1001654/crispr-cas9-gene-editing>

18. References:
White, M.K, Khalili, K, 2016, CRISPR?Cas9 and cancer targets: future possibilities and present challenges, Oncotarget, vol. 7, no. 11, pp. 123057, viewed on 21 May 2018,
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914286/>
Yeadon, J, 2018, Pros and Cons of ZNFS, TALENS and CRISPR/CAS, viewed 19th March 2018, <https://www.jax.org/news-and-insights/jax-blog/2014/march/pros-and-cons-
of-znfs-talens-and-crispr-cas>
Yi, L., Li, J., 2016, CRISPR-Cas9 therapeutics in cancer: promising strategies and present challenges, Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, vol. 1866, no. 2,
pp. 197-207, viewed on 20 May 2018, <https://www.sciencedirect.com/science/article/pii/S0304419X16300634 >
Yourgenome. (2016). What is CRISPR-Cas9?, viewed 19 May 2018, < https://www.yourgenome.org/facts/what-is-crispr-cas9 >
Yue, J, Du, Z, Zhou, F, Dong, P, Pfeffer, L. M, 2016 ‘Applications of CRISPR/Cas9 in Cancer Research’, Journal of Cancer Science and Research, vol. 1, no.1, pp. 104, viewed 19
May 2018, <https://www.omicsonline.org/open-access/applications-of-crsiprcas9-in-cancer-research-cmacd-1000103.pdf>
Zhuchi, T., Yan, S., li, X., Weili, Y. and Guo, X. (2015). CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases.
Researchgate, viewed 18 May 2018,
<https://www.researchgate.net/publication/280694279_CRISPRCas9_a_powerful_genetic_engineering_tool_for_establishing_large_animal_models_of_neurodegenerative_dis
eases?_sg=rD1i_Sourfvuhwgf4LXE1Ckpbr7Q51XhpdJ21Kikr9spLBuijq8gEgc9jgszDBLTrHMfSxo5tPdY-HqYjIn9OJ8RqbG8UTG53Q>