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PRACTICAL SKILLS > CMB2000/L13 CRISPR-CAS9 > Flashcards

CMB2000/L13 CRISPR-CAS9 Flashcards

1
Q

What does CRISPR-CAS9 stand for?

A

Clustered Regulatory Interspaced Short Palindromic Repeats and CRISPR Associated Proteins

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2
Q

What are the 3 components of CRISPR-CAS9?

A

Cas9 - protein component
crRNA - RNA component
tracrRNA - RNA component

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3
Q

What does the CRISPR-CAS9 complex do?

A

Cleaves invading DNA to prevent re-infection by viruses

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4
Q

How does CRISPR act as an adaptive immune regulator? (2)

A
  1. Invading DNA recognised and cut by Cas1-2 into fragments (protospacers)
  2. Protospacers integrated into CRISPR locus in bacterial genome
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5
Q

Describe what occurs upon viral reinfection with CRISPR. (4)

A

Transcription of protospacers activated
Bind to Cas9
Cas9/RAN duplex recruited to complementary sequence on invading DNA
Cas9 cuts DNA strands creating double strand break to prevent infection

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6
Q

Describe the structure of the CRIPSR locus. (4)

A

5’ transactivating RNA
Cas operon encoding Cas components
Identical repeat arrays
Spacers of invading DNA

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7
Q

What is the guide RNA (gRNA)?

A

Complex formed between transactivating RNA (tracrRNA) and protospacer/CRISPR RNA (crRNA)
Enables selective binding of Cas9 to invading DNA sequences

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8
Q

Explain how protospacer adjacent motifs (PAM) allow Cas9-mediated DNA cleavage.

A

Cas9 will not cut invading DNA without PAM site irrespective of Cas/gRNA binding

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9
Q

Describe the location of protospacer adjacent motifs.

A

2-8 base pair sequence 3-4 base pairs downstream of cut site

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10
Q

Give the 2 steps of Cas9 function in initial infection.

A

Protospacer aquisition
Spacer integration

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11
Q

Give the 4 steps of Cas9 function in re-infection.

A

Transcription
Binding to Cas9
Sequence comparison
Cleavage

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12
Q

How is gRNA modified for biomedical studies?

A

Linking cRNA and tracrRNA by adding linker loop
= composite gRNA

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13
Q

Give 2 properties of correct gRNA design.

A

Contains protospacer sequence (target sequence) upstream of PAM site
Should be selective to a single genome locus to avoid off target effects

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14
Q

If DNA is damaged by ionising radiation, double stranded breaks are repaired of one of two which mechanisms?

A

Homology-directly repair (HDR)
Non-homologous end-joining (NDEJ)

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15
Q

Describe how non-homologous end-joining (NHEJ) enables error-prone DNA repair. (3)

A

Introduces insertions or deletions into DNA
Impacts gene function
Often results in premature stop codons

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16
Q

Describe how homology-directed repair (HDR) enables precise DNA repair.

A

DNA precisely repaired using sister chromatid during S phase of cell cycle
Key to CRISPR knock-in studies

17
Q

Give the 3 steps to homology-directed repair after a double-strand break.

A

End resection
Strand invasion synthesis
Synthesis-dependent strand annealing

18
Q

Describe CRISPR-mediated gene knockout via NHEJ. (5)

A

Target Cas9:gRNA complex to gene of interest
DSB introduced
Cell repairs break via error-prone NHEJ
Indels introduced generate frameshift
Normal gene product not expressed

19
Q

Describe CRISPR-mediated gene knock-in via HDR. (5)

A

DSB introduced by Cas9-gRNA complex
Template introduced for use in repair
HDR template requires >60bp homology arms of either side of mutation/insert
PAM sites removed from HR template to prevent re-targeting of region
Inserts several kilobases possible

20
Q

Explain which receptor signalling is the main driver of prostate cancer. How is this useful in treatment?

A

Androgen receptors (AR)
Current treatment aims to inactivate AR by blocking ligand binding

21
Q

What is castrate-resistant prostate cancer?

A

Relapsed, incurable prostate cancer

22
Q

What 2 CRISPR-based studies are there for prostate cancer?

A

Generate Cas9-expressing prostate cancer cell line to knock-out AR
Create modified prostate cancer cell line to study function of aberrant forms of AR

23
Q

Describe what studies of androgen receptor variants have discovered so far. (2)

A

Alternative splicing principally involved in generation of AR-Vs in response to AR-targeting agents
Lack exons 4-8 encoding ligand-binding domain (LBD)
Loss of AR LBD creates constitutively active transcription factors refractory to enzalutamide
Expression of AR-Vs elevated in advanced disease

24
Q

Why is it difficult to study AR-Vs in isolation?

A

CWR22Rv1 cell line expresses both full length AR and AR-Vs
Full length AR (FL-AR) encoded by exons 1-8
AR-V7 lacks exons 4-8 giving shorter protein

25
Q

How is CRISPR hoped to cure prostate cancer? (2)

A

Block expression of full length AR
gRNA designed to exon 5 of AR
HDR template with point mutation encodes stop codon & stop formation of FL-AR

26
Q

Describe CWR22Rv1-AR-EK. (3)

A

First ever CRISPR knock-in prostate cancer cell line worldwide
Characterised how AR-Vs work by studying in isolation
Defined therapeutic vulnerability in prostate cancer cells expressing AR-Vs involving protein PARP1/2

27
Q

Give 3 considerations of cell therapy.

A

Efficacy of delivery
Regulatory guidelines
Mosaicism
Specificity
Immunogenicity
Germline vs somatic

28
Q

Describe ex-vivo delivery of CRISPR in the clinic. (4)

A

Remove cells from patient/donor
Edit genome
Screen/expand cell populations
Engraft cells back into patient

29
Q

Describe in-vivo delivery of CRISPR in the clinic. (2)

A

Package CRISPR/Cas in a delivery vehicle
Deliver to patient

30
Q

Describe the potential of CRISPR editing for HIV treatment.

A

Homozygous CCR5^32 donor gave stem cell transplant
Patient/recipient no longer required anti-viral therapy

31
Q

Describe how modified CCR5 results in a resistance to HIV infection.

A

Modified CCR5 means virus cannot bind to receptor and infect cells
32bp deletion results in frameshift and unstable protein

32
Q

Describe how successful CRISPR editing of CCR5 in vivo confers HIV-1 resistance.

A

Long term CCR5 disruption observed
CCR5 disrupted HSCs were able to reconstitute function immune system
Viral titre reduction and increase CD4+ T cells demonstrated HIV resistance

33
Q

Explain how CRISPR editing of CCR5 gene applies in humans.

A

Donor CCR5 wild-type gave stem cells
CRISPR used to disrupt CCR5 in donated HSCs
CCR5 ablated cells persisted for >19 months
Low amounts of modified cells means no HIV resistance

PRACTICAL SKILLS (18 decks)

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