An extensive presentation on the article: "Mechanism and Applications of CRISPR/ Cas-9-Mediated Genome Editing". DOI: https://doi.org/10.2147/BTT.S326422 CONTENTS: Components of CRISPR Mechanism of CRISPR/Cas 9 Genome editing Applications of CRISPR-Cas-9 Role in gene therapy Therapeutic Role Role in agriculture Role in gene silencing and activation Base Editors Prime Editors Challenges for CRISPR/Cas -9 application Recent advances Conclusion

1. Genome Editing and CRISPR-Cas 9
A presentation on the article “Mechanism and Applications of CRISPR/ Cas-9-Mediated Genome Editing”
Presentation prepared by Maliha Rashid
BS Biotechnology
Quaid-i-Azam University Islamabad
25 Dec 2021
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2. cOntent
• Components of CRISPR
• Mechanism of CRISPR/Cas 9 Genome editing
• Applications of CRISPR-Cas-9
1. Role in gene therapy
2. Therapeutic Role
3. Role in agriculture
4. Role in gene silencing and activation
• Base Editors
• Prime Editors
• Challenges for CRISPR/Cas -9 application
• Recent advances
• Conclusion
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3. Components of CRISPR Cas 9
1. Cas 9
• An Endonuclease enzyme
• Functions as a pair of molecular scissors
• Cut the target DNA sequence
2. Single-guide RNA
• Combination of tracrRNA (trans-activating crRNA) + crRNA (CRISPR
RNA)
• crRNA contains two main components
 Spacer sequence : Directs the complex to target DNA
 A region that binds to tracrRNA
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4. Components Explained…
• Recognition Lobe (REC) – REC1 and
REC2
• Nuclease Lobe (NUC) –RuvC, HNH and
PAM domain
CRISPR
associated
proteins or Cas-9
proteins
• CRISPR RNA (crRNA)
• Transactivating CRISPR RNA (tracrRNA)
Guide RNA
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5. 5

6. Mechanism of CRISPR/Cas-9
Mechanism involves three steps
1. Recognition
2. Cleavage
3. Repair
1. Recognition
• sgRNA directs Cas-9 and recognize the target sequence through its 5’crRNA.
• Cas-9 nucleases makes DSBs at a site 3 bp upstream to PAM
• Most common target for Cas-9 is 5’-NGG-3’ ( N can be any nucleotide)
• Once cas-9 found target site , it triggers local DNA melting followed by
formation of RNA-DNA hybrid
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7. 2. Cleavage
• Activation of Cas-9
• HNH domain cleaves the complementary strand , while RuvC domain
cleaves the non-complementary strand of target DNA
• Production of predominantly blunt-end DSBs.
3. Repair
• DSB is repaired by the host cellular machinery
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8. Applications of CRISPR/Cas-9
• Massive impact in the fields of medicine, agriculture, and
biotechnology.
1. Role in gene therapy
2. Therapeutic Role
3. Role in Agriculture
4. Role in gene activation and silencing
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9. Role in Gene Therapy
• More than 6000 genetic disorders have been known so far. But the
majority of the diseases lack effective treatment strategies.
• Gene therapy – replacing the defective gene with exogenous DNA
and editing the mutated gene at its native location.
• CRISPR/Cas-9 has been used in the treatment of sickle cell disease, β-
thalassemia, cystic fibrosis, and muscular dystrophy.
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10. CRISPR-Cas-9 in the treatment of SCD
• Treatment for SCD and β-thalassemia – in clinical trials.
• Two main approaches that CRISPR/Cas-9
1. direct repairing the gene of hemoglobin S
2. boosting fetal γ-globin – preferred approach
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11. 1. Bone marrow cells are removed from patients and the gene that
turns off fetal hemoglobin production, called B-cell Lymphoma 11A
(BCL11A) is disabled with CRISPR/ Cas-9.
2. Then, the gene-edited cells are infused back into the body.
3. Once this gene is disabled using CRISPR/ Cas-9, the production of
fetal hemoglobin containing γ-globin in the red blood cells will
increase, thereby alleviating the severity and manifestations of SCD
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12. CRISPR/Cas-9 for the treatment of cystic
fibrosis
• The genetic mutation of the cystic fibrosis transmembrane conductance regulator
(CFTR) gene decreases the structural stability and function of CFTR protein
leading to cystic fibrosis.
• In 2013, researchers culture intestinal stem cells from two cystic fibrosis patients
and corrected the mutation at the CFTR locus resulting in the expression of the
correct gene and full function of the protein.
• Since then, the potential utility of the application of CRISPR/Cas-9 for cystic
fibrosis was established.
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13. • Researchers corrected mutations that cause cystic fibrosis in
cultured human stem cells using a technique called prime
editing to replace the 'faulty' piece of DNA with a healthy
piece.
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15. Duchenne muscular dystrophy (DMD)
• caused by a mutation in the dystrophin gene and
characterized by muscle weakness
• DMD has been successfully corrected by CRISPR/Cas-9
in patient-induced pluripotent stem cells that restored
the expression of DMD gene
• the treatment available for DMD remains supportive
rather than curative
• several therapeutic approaches - gene therapy, cell
therapy, and exon skipping – current investigation
• Deletion/excision of intragenic DNA and removing the
duplicated exon by CRISPR/Cas-9 – new approach
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17. Therapeutic Role of CRISPR/Cas-9
• CRISPR-Cas-9
therapy – first
human trial was
conducted to
treat patients
with refractory
lung cancer.
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18. Role in HIV Treatment
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19. Role in Agriculture
Genetically modify foods to improve:
1. nutritional value
2. increase their shelf life
3. make them drought-tolerant
4. enhance disease resistance
Three ways that CRISPR is solving the world’s food crisis:
1. restore food supplies
2. help plants to survive in hostile conditions
3. could improve the overall health of the plants
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21. Role in Gene Activation and Silencing
• Activate or repress genes (by the use of dCas-9)
• Visualize and pinpoint where a specific gene of interest
is present in the genome – subcellular localization by
fusing with GFP proteins
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22. Schematic of the CRISPRi approach. Left, Cartoon representation of the CRISPR-mediated regulation of gene
expression. The wild-type Cas9 protein binds to the sgRNA and forms a protein–RNA complex. Once Cas9–sgRNA
complex binds to specific DNA target adjacent to PAM, it leads to the cleavage of the target DNA due to the nuclease
activity of the Cas9 protein. Right, Cartoon depicting the CRISPRi-mediated interference of gene expression caused by
nuclease-deficient dCas9. The nuclease-deficient dCas9 contains two substitutions in the nuclease domains (D10A and
H840A, blue dots), and thus lose the endonuclease activity. If the target DNA sequence locates inside an open reading
frame, the dCas9–sgRNA–DNA complex will block the movement of RNAP and subsequent transcription elongation,
resulting in transcription inhibition of the target gene
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23. Base Editors
1. cytosine base editor (CBE)
2. adenine base editor (ABE)
• The CBE is a type of base editor composed of cytidine deaminase
fused with catalytically deficient or dead Cas-9 (dCas-9).
• However, the target range of the CBE base editor is still restricted by
PAM sequences containing G, T, or A bases.
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24. • The ABE uses adenosine deaminase fused to dCas-9 to correct the
base-pair change from adenosine (A) to guanosine (G).
• Overall, single-base editing through the fusion of dCas-9 to cytidine
deaminase or adenosine deaminase is a safe and efficient method to
edit point mutations.
• But both base editors can only fix four-transition mutations (purine to
purine or pyrimidine to pyrimidine).
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25. Prime Editors
To overcome this shortcoming, the most recent member of the CRISPR
genome editing toolkit called Prime Editor (PE) has been developed to
extend the scope of DNA editing beyond the four types of transition
mutations.
PE contains
1. Cas-9 nickase fused with engineered reverse transcriptase
2. multifunctional primer editing guide RNA (pegRNA)
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26. Components of Prime Editors and their
Functions
1. The pegRNA recognizes the target nucleotide sequence
2. the Cas-9 nickase cuts the non-complementary strand of DNA three bases
upstream from the PAM site, exposing a 3ʹ-OH nick of genomic DNA
3. The reverse transcriptase then extends the 3ʹ nick by copying the edit
sequence of pegRNA.
• Hence, PE not only corrects all 12 possible base-to-base transitions, and
transversion mutations but also small insertion and deletion mutations in
genetic disorders. 26

27. Challenges for CRISPR/Cas-9 Application
• Immunogenicity, lack of a safe and efficient delivery system to the
target, off-target effect, and ethical issues have been the major
barriers to extend the technology in clinical applications.
• How to detect and reduce the immunogenicity of Cas-9 protein.
• Three methods of delivering the CRISPR/Cas-9 complex into cells,
1. physical
2. chemical
3. viral vectors
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28. Physical Methods Of Delivering CRISPR/ Cas-9
• Electroporation -- the main limitation of this method is that it causes
significant cell death
• Microinjection -- this method also has several disadvantages such as
cell damage
• Hydrodynamic injection -- it has not yet been used in clinical
applications due to possible complications
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29. Chemical Methods
• The chemical methods of
CRISPR/Cas-9 delivery involves
lipid and polymer-based
nanoparticles.
• Lipid nanoparticles, polymer-
based nanoparticles can also
transverse the complex in the
membrane through
endocytosis.
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30. Viral Vectors
• The limited virus cloning capacity and the large size of the Cas-9
protein remain a major problem
• The off-target effect can possibly cause harmful events such as
sequence mutation, deletion, rearrangement, immune response, and
oncogene activation, which limits the application of the CRISPR/Cas-9
editing system for therapeutic purposes
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31. Recent Advances
To mitigate the possibility of CRISPR/Cas-9 off-target effect, several
strategies have been developed.
• Optimization of sgRNA, modification of Cas-9 nuclease, utilization of
other Cas-variants, and the use of anti-CRISPR proteins.
• Selecting and designing an appropriate sgRNA for the targeted DNA
sequence is an important first step to reduce the off-target effect
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32. • It has been reported that the use of the inactivated RuvC domain of
Cas-9 with sgRNA can reduce the off-target effect by 100 to 1500
times.
• the use of multicomponent Class I CRISPR proteins, such as CRISPR/
Cas-3 and CRISPR/Cas-10 provides better genome editing efficiency
than Cas-9
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33. Summary
CRISPR/Cas-9 gene editing is adopted from acquired immunity in prokaryotes and consists of two
elements:
• Guide RNA used to locate (bind) the target DNA to be edited and Cas-9, a protein that essentially
cuts the DNA at the location identified by guide RNA
• The fundamental part of the CRISPR/Cas-9 gene-editing process is the identification of the target
gene that determines the phenotype of interest and the designing the guide RNA.
• This process becomes a new era in molecular biology and has countless roles ranging from basic
molecular research to clinical applications.
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34. THANKS
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