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PATH3309 > 15 - CRISPR (PATH08) > Flashcards

15 - CRISPR (PATH08) Flashcards

1
Q

What does CRISPR stand for

A

Clustered Regularly Interspaced Short Palindromic Repeats

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2
Q

CRISPR

A
  • Array of short repeated sequences separated by spacers with unique sequences
  • Found on both chromosomal and plasmid DNA in bacteria
  • Bacteria capture snippets of DNA from invading viruses
  • CRISPR system allows the bacteria to “remember” viruses (or closely related ones)
  • Bacteria use Cas9 (or other Cas enzyme) to cut the DNA and disable the virus
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3
Q

Three main components of CRISPR-Cas9

A
  • Cas9
  • Guide RNA (gRNA): composed of crRNA and tracrRNA
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4
Q

crRNA

A
  • CRISPR RNA (“spacer”)
  • sequence-specific, recognizes target sequence
  • Adjacent to PAM
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5
Q

PAM

A
  • Protospacer Adjacent Motif
  • Distinguishes self from non self
  • 3-5 nucleotides in length
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6
Q

tracrRNA

A
  • trans-activating crRNA
  • scaffold for Cas9
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7
Q

How are components delivered to cells

A
  • Using plasmids, or synthesized and delivered directly as ribonucleoprotein (RNP) complexes
  • RNP has rapid turnover and therefore preferred
  • Can be delivered as mRNA
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8
Q

What does Cas9 absolutely require in the target DNA in order to induce DNA cleavage

A

PAM

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9
Q

How is Cas9 directed to target site

A

gRNA

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10
Q

Two main pathways to repair DNA damage in
eukaryotic cells

A
  • Non-Homologous End Joining (NHEJ)
  • Homology Directed Repair (HDR)
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11
Q

NHEJ

A
  • Typically leads to deletion (or insertion) of nucleotides
  • Can cause frameshift and inactivation of gene (nonsense mutation)
  • Operates through all stages of cell cycle
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12
Q

HDR

A
  • Used to make specific changes by providing a repair template
  • Less efficient (25% vs 75% for NHEJ)
  • Predominantly occurs in S/G2 phase
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13
Q

Delivery methods of CRISPR

A
  • Different types of cells and tissues are more receptive to different delivery methods
  • AAV
  • Microinjection
  • Electroporation
  • Lipofection
  • Nanoparticles
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14
Q

Expression plasmid advantages and disadvantages

A
  • Cheap, expandable
  • BUT Genomic integration issues, and prolonged expression
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15
Q

Viral vectors advantages and disadvantages

A
  • High delivery efficiency, expandable
  • BUT genomic integartion issues, must cas variants too big for viral vector
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16
Q

In vitro transcribed (IVT) gRNA and Cas9 mRNA advantages and disadvantages

A
  • Non integrating
  • BUT Low stability of RNA, potential immunogenic effects, delay in translation
17
Q

RNP complex advantages and disadvantages

A
  • Non integrating, immediate acting
  • BUT expensive and single use
18
Q

Nanoparticle delivery

A
  • E.g. lipid nanoparticles
  • Can reduce Cas9 recognition and clearance by
    immune cells
  • Used successfully in vaccines
  • Can use conjugates to improve delivery
19
Q

Advantages of lipid nanoparticles

A
  • Biodegradable
  • Low immunogenicity
  • Large cargo capacity
20
Q

CRISPR for gene activation or repression

A
  • Catalytically inactivated Cas9, has no cleavage activity
  • ‘dead’ Cas9 (dCas9)
21
Q

dCas9

A

dCas9 can be fused to various effectors that enable
activation or repression of target loci

22
Q

Applications that do not involve DNA cleavage (NHEJ and HDR)

A
  • Transcriptional activation
  • Transcriptional repression
  • Epigenetic modification
  • Single base editing
23
Q

Utility of CRISPR for research and clinical applications

A
  • Investigate function of new genes
  • Correct underlying mutation or delete mutant copy of gene
  • As a therapy to correct DNA level mutations (permanent cure)
24
Q

iPSCs

A
  • Induced pluripotent stem cells
  • High self-renewal rate and can differentiate into
    almost all cell types
25
Q

Monogenic/Mendelian diseases

A
  • CF
  • Haemophilia
  • Sickle cell
  • DMD
26
Q

CF

A
  • Autosomal recessive disease caused by mutations in CFTR gene
  • Build up of mucus in lungs and other organs leaving to severe impairment of lung function
27
Q

Most common mutation in CF

A

3bp in frame deletion that impairs protein folding, maturation and transport to surface of cell

28
Q

Major challenge of CF therapy

A
  • Targeting basal (stem) cells would be the most likely to sustain long term functional restoration
  • Airway epithelium is primarily made up of non-dividing cells, which limits efficient HDR repair
  • Difficult to transduce basal cells (stem cells that generate airway epithelial cells)
29
Q

Sickle cell disease (SCD)

A
  • Caused by specific point mutation in gene that encodes beta chain hemoglobin
  • Recessive disorder
  • Red blood cells (RBCs) assume ‘sickled’ shape, cells clump together and stick to small blood vessels
  • Causes anemia, stroke, death
30
Q

SCD therapy

A
  • Shortly after birth, babies stop producing fetal hemoglobin (HBG; gamma hemoglobin), and switch over to adult hemoglobins (HBB; beta hemoglobin)
  • Rare individuals continue to make high levels of fetal hemoglobin (HbF) throughout their lives are still entirely healthy; hereditary persistence of fetal hemoglobin (HPFH)
  • Rare individuals with SCD who also have HPFH have an extremely mild version of SCD that provides protection against sickling
  • Gene editing to increase levels of HbF in RBCs of people with SCD
31
Q

BCL11A

A

Disruption of enhancer reduces BCL11A expression, and induces expression of fetal γ-globin

32
Q

Clinically approved drug for SCD

A

Casgevy

33
Q

DMD

A

Severe degenerative muscular disease caused by loss-of-function mutations in the dystrophin gene (DMD), located on the X chromosome

34
Q

Hemophilia B

A
  • Inherited disease caused by mutations in the coagulation factor IX (FIX) gene
  • X-linked recessive disorder
35
Q

Limitations of CRISPR

A
  • Delivery method
  • Off target effects
  • Immune response
  • Time and cost
  • Ethical considerations
36
Q

Ethical concerns

A
  • Eugenics
  • Misapplication
  • Inequitable access
  • Regulation
  • Embryo modification

PATH3309 (26 decks)

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