Assignment 2

Team F4

Chance of survival: CRISPR gene therapy

https://youtu.be/JZLaA0BdXKc?si=Vs0TKMykoYZ_653q
Introduction
Genetic disease is the fundamental cause of many long term illness which
scientists and doctor were struggling to understand and cure them.

● Maternal genetic disorders: Down Syndrome, Sickle cell anemia.

● After birth: Cancer ( ⅓ of the cause of death in HK)

With advanced knowledge in Genetics, many genetic disorder are still unavoidable
nor curable. The only advice for the parents-to-be are to avoid having a baby.
CRISPR is their way out!

(Uddin et al., 2020)

 RNA guided gene editing→Precision
(Uddin et al., 2020)

Target Recognition HDR: replace or introduce

CRISPR-Cas9 System The gRNA binds to the Cas9 protein,
Cas9 protein and a guide RNA(gRNA). forming a complex. This complex then specific DNA sequence at target site
The gRNA is designed to be searches for a DNA sequence and
complementary to a specific target matches the gRNA, creates a double-strand
sequence in the DNA. break at that location in the DNA.
● DNA with homology to the
sequence flanking the DSB site.
● gRNA Design: ● Acted as a template for DNA
repair→contain specific changes
○ a CRISPR RNA (crRNA), which is complementary
or insertions
to the target DNA sequence
○ a trans-activating CRISPR RNA (tracrRNA), DNA Repair & Editing
which helps the gRNA bind to the Cas9 protein. ● NHEJ(Nonhologous End Joining): MUTATION!
This pathway repairs the break by directly joining
the two ends of the DNA.

● Cas9 Protein: The Cas9 protein is an enzyme ● HDR(Homology-directed repair):

that acts as a molecular scissors. ACCURATE! ✓template containing the desired
genetic changes. The cell's repair machinery uses
the template to replace the damaged DNA sequence

Delivery
viral vectors or non-viral
methods
(e.g: lipid nanoparticles).
(Uddin et al., 2020)
(Uddin et al., 2020)
Delivery of CRISPR Therapy
recognizes anitigen to
mediate immune response
(Li et al., 2020)

Types of CRISPR cargo:

Cancer Immunotherapy
1. DNA 2. RNA 3. Protein

CRISPR vehicles: (Glass et al.,2018)

Application
Adeno-Associated Viral Vectors (AAV)
Electroporation NY-ESO-1 antigen:
pros: - high efficiency of delivery →opening transient pores in the membrane
cons: - risk of immunotoxicity & insertional of cells via high-voltage electrical currents, frequently found on the
oncogenesis allowing CRISPR cargo to gain entry
● small in capacity limiting size of cargo cons: surface of relapsed tumors
● ↑incidence of OTEs - electric current can be toxic
- can lead to permanent permeabilization → serve as therapeutic target
Limitation of CRISPR gene therapy
❖ Off-target effects (OTEs)
➢ endanger the patient's health.
■ lack of complete specificity, genetic variability

❖ Immune responses
➢ Misinterpreted as foreign →trigger immune response,
■ neutralise the therapy / result in unfavourable effects.
❖ Complex genetic disorders
(Uddin et al., 2020)
■ ✔potential in the treatment of single-gene illnesses
■ more complicated genetic disorders present difficulties.
■ Numerous genes and intricate interactions are involved in a number of disease.
❖ Limited targetable genes
➢ difficult to target many genes
■ functions are still often unclear
■ several genes may have multiple functions in various tissues / developmental stages
■ necessitating careful thought before choosing a therapeutic target.
 Conclusion
● Precision of CRISPR
○ gRNA design and Cas9 protein: target a specific region
○ HDR : replace or introduce specific DNA sequence at target site

● Delivery of CRISPR and its application

○ using DNA RNA or Protein as CRISPR cargo
○ non viral/ viral vectors
○ application: cancer treatment

● Limatation of CRISPR gene therapy

○ Off-target effects (OTEs)
○ Immune responses
Advanced technology → ✖limitation
○ Complex genetic disorders Improvement →↑successful rate
○ Limited targetable genes In future: More patient healed
Workload

Name UID Work Done(page) Percentage%

Cheung Lap Yan 3036221210 1,2,3,6 35

Chui Long Ching 3036058699 6,8 20

Eunice Kwan 3036220943 5,7,8 25

Tam Pak Ho 3036218445 4,8 20

Bibliography
❏ (Main source) Pak, Ekaterina. 2014. “CRISPR: A Game-Changing Genetic Engineering Technique.” Science in the
News. Harvard University. July 31, 2014.
https://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/.
❏ Uddin F, Rudin CM and Sen T. 2020. “CRISPR Gene Therapy: Applications, Limitations, and Implications for the
Future.” Frontiers in Oncology 10 (1387). https://doi.org/10.3389/fonc.2020.01387.
❏ Glass Z, Lee M, Li Y, Xu Q. 2018. "Engineering the Delivery System for CRISPR-Based Genome Editing." Trends
Biotechnol. 2018 Feb. https://pubmed.ncbi.nlm.nih.gov/29305085/
❏ Li J. 2020. "How CRISPR Is Changing Cancer Research and Treatment." National Cancer Institute. 27 JUL 2020.
https://www.cancer.gov/news-events/cancer-currents-blog/2020/crispr-cancer-research-treatment
❏ Manuel M Vicente et al. Front Cell Dev Biol. 2021. “The Off-Targets of Clustered Regularly Interspaced Short
Palindromic Repeats Gene Editing” The Off-Targets of Clustered Regularly Interspaced Short ... - PubMed