15 - CRISPR (PATH08) Flashcards
What does CRISPR stand for
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR
- 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
Three main components of CRISPR-Cas9
- Cas9
- Guide RNA (gRNA): composed of crRNA and tracrRNA
crRNA
- CRISPR RNA (“spacer”)
- sequence-specific, recognizes target sequence
- Adjacent to PAM
PAM
- Protospacer Adjacent Motif
- Distinguishes self from non self
- 3-5 nucleotides in length
tracrRNA
- trans-activating crRNA
- scaffold for Cas9
How are components delivered to cells
- Using plasmids, or synthesized and delivered directly as ribonucleoprotein (RNP) complexes
- RNP has rapid turnover and therefore preferred
- Can be delivered as mRNA
What does Cas9 absolutely require in the target DNA in order to induce DNA cleavage
PAM
How is Cas9 directed to target site
gRNA
Two main pathways to repair DNA damage in
eukaryotic cells
- Non-Homologous End Joining (NHEJ)
- Homology Directed Repair (HDR)
NHEJ
- Typically leads to deletion (or insertion) of nucleotides
- Can cause frameshift and inactivation of gene (nonsense mutation)
- Operates through all stages of cell cycle
HDR
- Used to make specific changes by providing a repair template
- Less efficient (25% vs 75% for NHEJ)
- Predominantly occurs in S/G2 phase
Delivery methods of CRISPR
- Different types of cells and tissues are more receptive to different delivery methods
- AAV
- Microinjection
- Electroporation
- Lipofection
- Nanoparticles
Expression plasmid advantages and disadvantages
- Cheap, expandable
- BUT Genomic integration issues, and prolonged expression
Viral vectors advantages and disadvantages
- High delivery efficiency, expandable
- BUT genomic integartion issues, must cas variants too big for viral vector
In vitro transcribed (IVT) gRNA and Cas9 mRNA advantages and disadvantages
- Non integrating
- BUT Low stability of RNA, potential immunogenic effects, delay in translation
RNP complex advantages and disadvantages
- Non integrating, immediate acting
- BUT expensive and single use
Nanoparticle delivery
- E.g. lipid nanoparticles
- Can reduce Cas9 recognition and clearance by
immune cells - Used successfully in vaccines
- Can use conjugates to improve delivery
Advantages of lipid nanoparticles
- Biodegradable
- Low immunogenicity
- Large cargo capacity
CRISPR for gene activation or repression
- Catalytically inactivated Cas9, has no cleavage activity
- ‘dead’ Cas9 (dCas9)
dCas9
dCas9 can be fused to various effectors that enable
activation or repression of target loci
Applications that do not involve DNA cleavage (NHEJ and HDR)
- Transcriptional activation
- Transcriptional repression
- Epigenetic modification
- Single base editing
Utility of CRISPR for research and clinical applications
- Investigate function of new genes
- Correct underlying mutation or delete mutant copy of gene
- As a therapy to correct DNA level mutations (permanent cure)
iPSCs
- Induced pluripotent stem cells
- High self-renewal rate and can differentiate into
almost all cell types
Monogenic/Mendelian diseases
- CF
- Haemophilia
- Sickle cell
- DMD
CF
- 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
Most common mutation in CF
3bp in frame deletion that impairs protein folding, maturation and transport to surface of cell
Major challenge of CF therapy
- 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)
Sickle cell disease (SCD)
- 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
SCD therapy
- 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
BCL11A
Disruption of enhancer reduces BCL11A expression, and induces expression of fetal γ-globin
Clinically approved drug for SCD
Casgevy
DMD
Severe degenerative muscular disease caused by loss-of-function mutations in the dystrophin gene (DMD), located on the X chromosome
Hemophilia B
- Inherited disease caused by mutations in the coagulation factor IX (FIX) gene
- X-linked recessive disorder
Limitations of CRISPR
- Delivery method
- Off target effects
- Immune response
- Time and cost
- Ethical considerations
Ethical concerns
- Eugenics
- Misapplication
- Inequitable access
- Regulation
- Embryo modification
