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Molecular Biology of Disease > Genome editing (CRISPR) > Flashcards

Genome editing (CRISPR) Flashcards

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1
Q

What is genome editing?

A

A type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases.

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2
Q

What do nucleases do?

A

Cut the DNA double helix at a particular recognition sequence. The cut can be repaired in a way that introduces mutations.

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3
Q

How is genome editing used in drug development?

A

Allows target validation; helps confirm a molecule is going to be a good drug target. Gene KO replicates what a drug would do.

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4
Q

How is genome editing used in animal models?

A

Introduce a mutation thought to be associated with a disease and observe whether the disease phenotype occurs in the animal.

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5
Q

How is genome editing used in the food industry?

A

Making crops resistant to pathogens or with increased yields.

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6
Q

What is gene surgery?

A

Using genome editing in cells from patients to correct a defect (cells edited in vitro and then injected back into patient, or nucleases injected and act in vivo).

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7
Q

Why is engineering zinc finger DNA binding proteins (ZFs) difficult?

A

ZFs only bind 3 nucleotide sequences. Design was hard because binding a string of ZFs on a longer target sequence interfered with the specificity of each ZF. Also it was hard to find a ZF in vitro that behaved how you wanted to base your design off.

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8
Q

Which proteins have been used for gene editing?

A

1990s - 2012: zinc fingers
2010 - 2014: TAL effectors
2013 onwards: CRISPR

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9
Q

Why was synthesising TAL effector DNA binding proteins hard?

A

Each nucleotide required a separate TAL effector domain for binding, and synthesising all these domains together was complex. No interference though!

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10
Q

Why was CRISPR/Cas9 design so easy?

A

CRISPR involves RNA sequences recognising DNA sequences, and the interactions between RNA and DNA are well characterised so easy to design and synthesise. The same Cas9 enzyme can be used for any editing.

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11
Q

What is the CRISPR/Cas9 genetic editing process?

A
  • Cas9 nuclease binds a CRISPR RNA sequence (gRNA). This is typically generated through phage genomes that got incorporated into bacterial DNA.
  • Cas9 binds a PAM and unwinds the double helix.
  • The gRNA searches for homology within this sequence.
  • Sufficient homology causes a conformational change in the Cas9 that activates its endonuclease activity.
  • 2 domains of Cas9 (1 for each DNA strand) cut the DNA - forms a DSB.
  • If DSB is immediately repaired the process can repeat.
  • An exonuclease cleaves a few nts from the cut site.
  • Repair results in a small insertion or deletion mutation.
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12
Q

What is the protospacer adjacent motif (PAM)?

A

An NGG sequence in the genomic DNA 3-4 nts downstream of a potential CRISPR/Cas9 target site. The Cas9 nuclease binds it.

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13
Q

What functions does the Cas9 enzyme have?

A
  • Helicase (unwinds DNA)
  • Endonuclease (cuts DNA)
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14
Q

Is CRISPR/Cas9 more efficient than ZF or TAL effectors?

A

Yes - it has much higher editing rates. This allows experiments to be scaled up.

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15
Q

Can we quantify what proportion of DNA breaks result in mutations?

A

No - this is dependent on the sequence being cut and and exonucleases and repair factors interact with it.

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16
Q

How can we change the CRISPR/Cas9 system to have a different function?

A

Inactivate Cas9’s endonuclease domains then tether a domain with different action to the Cas9.

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17
Q

What are alternative domains fused to the CRISPR/Cas9 system?

A
  • Transcriptional activator / repressor (changes gene expression)
  • Fluorescent protein (allows visualisation of movements of regions of the genome within the nucleus)
  • DNA looping factor (changes the topology of the DNA)
  • Cytidine deaminase (also introduces mutations - related to base editing)
  • Reverse transcriptase
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18
Q

Why do DSBs in the genome need to be repaired?

A

They can be highly toxic and lead to genetic disease.

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19
Q

What is non-homologous end joining (NHEJ)?

A
  • Exonucleases bind the 3’ ends of the broken strands and digest in the 3’ to 5’ direction.
  • Strands are joined but a different base sequence (insertion or deletion) is the result.
  • Frame shift often results in a premature stop codon.
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20
Q

How does the position of the mutation affect gene function?

A
  • Near the 5’ end usually results in gene KO.
  • Near the 3’ end usually results in change in gene function.
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21
Q

Why can’t we use CRISPR/Cas9 to repair a specific mutation in a patient?

A

Because the outcome of the cut is not predictable; you get a range of insertions and deletions with the same treatment.

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22
Q

What is homology directed repair (HDR)?

A
  • Exonucleases bind the 3’ ends of the broken strands and digest in the 3’ to 5’ direction.
  • A piece of DNA that is (almost) homologous to the broken strand is used as a template.
  • The repaired sequence matches that of the template (induces the change present in the template, ie useful for gene repair).
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23
Q

How do we encourage HDR (compared to NHEJ) when using CRISPR/Cas9?

A

Provide a high concentration of a template molecule (ssDNA).

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24
Q

How is the gene repair pathway (NHEJ or HDR decided)?

A

The pathways always exist in competition, but which is favoured depends on the state of the cell.
Usually NHEJ is favoured, but in S phase HDR is favoured. We are researching how to promote HDR over NHEJ.

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25
Q

What are the advantages of somatic gene therapy?

A
  • The genetic changes can be targeted to a subset of cells in the treated individual.
  • Fewer ethical concerns.
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26
Q

What are the disadvantages of somatic gene therapy?

A
  • Difficult for inaccessible tissues / cells (may require delivery of Cas9/gRNA/donor template in situ).
  • Repeated doses of therapy may be required to maintain clinical benefit.
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27
Q

What are the advantages of germline gene therapy?

A
  • Changes affect all cells of the body.
  • Changes can be transmitted to offspring.
  • Genetic changes can be introduced in a single treatment in IVF (zygote edited).
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28
Q

What are the disadvantages of germline gene therapy?

A
  • Issues of consent.
  • Almost always alternatives that don’t require gene modification.
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29
Q

What are technological challenges with using genome editing technologies to treat genetic diseases?

A
  • Delivering the necessary molecules to the right cells.
  • Avoiding off-target mutagenesis.
  • Achieving the desired genetic change at the intended target site.
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30
Q

What needs to be considered when determining how to deliver molecules to target cells?

A
  • Which cells need to be targetted? (Dependent on disease).
  • Can these cells be edited ex vivo? (Editing is easier ex vivo).
  • Do target cells persist in the body or die after a few days? (Single treatments are less invasive).
  • In what format will the editing molecules be delivered? (DNA / RNA / protein).
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31
Q

What format can a donor template be delivered in?

A

DNA.

32
Q

What formats can a gRNA be delivered in?

A

DNA or RNA.

33
Q

What formats can the Cas9 nuclease be delivered in?

A

DNA, RNA or protein.

34
Q

What are advantages of delivering gRNA and Cas9 in DNA form?

A
  • Long lived in cells.
  • Compatible with many vector systems.
35
Q

What are disadvantages of delivering gRNA and Cas9 in DNA form?

A
  • Can integrate into the genome (off target mutagenesis).
  • Can activate anti-viral responses in primary cells, resulting in death of the cells.
36
Q

What are advantages of delivering gRNA and Cas9 in RNA form?

A
  • Less likely to integrate into the genome (would need to be reverse transcribed).
  • Less likely to activate anti-viral response, so more effective in primary cells.
37
Q

What are disadvantages of delivering gRNA and Cas9 in RNA form?

A
  • Short persistence in cells.
38
Q

What are advantages of delivering gRNA and Cas9 in ribonucleoprotein form?

A
  • Will not integrate into the genome.
39
Q

What are disadvantages of delivering gRNA and Cas9 in ribonucleoprotein form?

A
  • Shortest persistence in cells, so least time for genome editing.
40
Q

Which cell types are the easiest to target with genetic editing treatments?

A

Cells that are accessible:
- Blood cells (RBCs, HSCs, T cells; edited to fight cancer)
- Urinary tract cells (accessible in situ)
- Eye cells (accessible in situ)
- Liver cells (molecules injected into the blood are efficiently taken up by the liver)

41
Q

Why is ex vivo editing easier?

A
  • Cell environment can be controlled to favour desired editing outcomes.
  • Cells with desired editing outcomes can be selected (ideally stem cells).
  • Cells with off target mutations can be excluded (quality control).
42
Q

Why are hematopoietic stem cells (HSCs) ideal for editing?

A
  • Can be isolated from blood
  • Can be cultured ex vivo (self renew)
  • Give rise to all other blood cell types
  • Can all be ablated with drugs so they can be replaced with edited cells
  • Persist in vivo for decades.
43
Q

Which cell types are the hardest to target with genetic editing treatments?

A
  • Lung (rapid cell turnover)
  • Heart (inaccessible)
  • Prostate (inaccessible)
  • Brain (inaccessible)
44
Q

What is transfection?

A

Encapsulating molecules in lipid particles that can traverse the cell membrane.

45
Q

How do we get molecules to enter cultured cells?

A
  • Transfection
  • Electroporation
46
Q

What is electroporation?

A

Applying electric currents to cells to open up pores in the membrane to allow molecules through.

47
Q

What extra challenge does editing in tissues instead of in cultured cells present?

A

Molecules must find and exclusively enter the right cell type in the body.

48
Q

How are gene therapies targeted to specific cell types?

A
  • Using viral vectors e.g. AAV because they evade cellular defences and deliver genetic material into human cells.
  • Using nanoparticles because they can be designed to target specific cell types.
49
Q

Why is AAV a popular viral vector?

A
  • Not highly immunogenic.
  • Exists as an extrachromosomal molecule (endogenous genes not disrupted).
50
Q

What is an issue with using AAV as a vector?

A

It has a small packaging capacity so some systems can’t fit into it.

51
Q

What improvements to vectors are being investigated?

A
  • Holding more cargo
  • Additing cell specificities
  • Lowering toxicity
52
Q

Why may CRISPR/Cas9 treatment generate off-target mutagenesis?

A

In bacteria, Cas9 tolerates slight mismatches between CRISPR RNA and phage DNA because phages can evolve quickly.

53
Q

How have we reduced Cas9 off-target mutagenesis?

A
  • Careful gRNA design (screen against target genome during design process).
  • Preclinical testing of the gRNA in cultured cells to check toxicity and realistic mutational profiles.
  • Engineering of Cas9 to reduce tolerance for mismatches.
54
Q

Why does off-target mutagenesis need to be minimised?

A
  • Increased frequency of mutations can promote cancer e.g. with mutations that inactivate TSGs or activate oncogenes.
  • Excess DSBs can activate the p53 pathway which increases chance of cell death.
55
Q

What is the more simple use of CRISPR/Cas9 editing?

A

Knockout of a gene with abnormal function - NHEJ resulting in deletions of variable lengths that disrupt ORFs is suitable.

56
Q

What is the more difficult use of CRISPR/Cas9 editing?

A

Correction of a non-functional gene - use of a donor template to stimulate HDR is more technically challenging as more precision is required. Typically <5% of DNA breaks are perfectly repaired with donor templates. This needs improving!

57
Q

Which points in the cell cycle is NHEJ active?

A

All.

58
Q

Which points in the cell cycle is HDR active?

A

S / G2 / M (after chromosome duplication, ie a template for repair is available).

59
Q

Which cells are precise edits (HDR) easier to achieve in?

A

Those that are passing through the cell cycle frequently. But lots of our cells are terminally differentiated!

60
Q

How can we target editing to one (mutated) allele? (Dominant disease).

A

Design the gRNA to only be complementary to the mutant allele (‘allele-specific’).

61
Q

How might CRISPR/Cas9 be used for genetic editing without DSBs?

A

Through Cas9 fusion proteins carrying out activities e.g. prime editing and base editing.
Allows precise editing in non-dividing cells without HDR.

62
Q

How is CRISPR/Cas9 base editing achieved?

A
  • Cas9 nickase is used (only 1 DNA strand cleaved).
  • Cas9 nickase is fused to cytidine deaminase.
  • C is stably edited to U, which is T when replicated. The other strand is converted from G to A after replication.
63
Q

What are disadvantages of base editing?

A
  • Don’t have single nt capacity yet; every C in the cytidine deaminase range gets converted to U. This introduction of new mutations can be problematic, even if the target mutation is fixed.
  • Not all base modifications are possible.
  • No scope for reversing insertions and deletions as no DSBs are being made.
64
Q

What are advantages of base editing?

A
  • Does not require DSBs therefore there is less activation of DDR and less potential for off target mutations.
  • Does not require active HDR, so can work in non-cycling cells.
  • More control over DNA repair outcome; rates of perfect editing higher than Cas9 with HDR.
65
Q

How is CRISPR/Cas9 prime editing achieved?

A
  • Cas9 nickase is used (1 DNA strand cleaved).
  • Cas9 nickase is fused to a reverse transcriptase.
  • gRNA is swapped for pegRNA.
  • 3’ end of the ssDNA is hybridised to pegRNA (only to end of pegRNA).
  • Reverse transcription of the pegRNA.
  • DNA contains altered sequence encoded in pegRNA.
  • DNA repaired.
66
Q

What is a disadvantage of prime editing?

A
  • pegRNA and reverse transcriptase are too large to fit in an AAV vector.
67
Q

What is gene therapy?

A

Treatment or prevention of a disease by altering the patient’s genes:
- KO of faulty gene
- Repair of faulty gene

68
Q

Why is CRISPR being looked at for use in gene therapies?

A

It will improve the effectiveness of the therapies.

69
Q

How many CRISPR-mediated therapies are in clinical trials?

A

> 80

70
Q

Why are drug companies disincentivised from finding research for drugs for rare genetic disorders?

A

They won’t make much money off it because not many people need the drug.

71
Q

Why is there not much research into rare genetic disease treatments?

A

There is an economic block - the drug companies won’t fund it.

72
Q

What is CAR-T cell therapy?

A

T cells have a synthetic Chimeric Antigen Receptor expressed as a transgene, which gives the T cell the ability to recognise a particular molecule e.g. something highly expressed in a cancer. This leads to immune response against and destruction of the cell expressing this molecule.

73
Q

What do antigen receptors allow T cells to do?

A

Recognise self from non-self.

74
Q

Which antigen are CAR-T cells currently being developed for?

A

CD19. Commonly expressed on B cell tumours (leukemias and lymphomas). We know the receptor protein gene.

75
Q

Which delivery system is unreliable at facilitating CAR-T receptor expression?

A

Retroviruses - the virus integrated at different points in the genome and expression of the receptor was variable. The integration location is important but difficult to control in this case.

76
Q
A

Molecular Biology of Disease (4 decks)

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