CMB2000/L13 CRISPR-CAS9 Flashcards
What does CRISPR-CAS9 stand for?
Clustered Regulatory Interspaced Short Palindromic Repeats and CRISPR Associated Proteins
What are the 3 components of CRISPR-CAS9?
Cas9 - protein component
crRNA - RNA component
tracrRNA - RNA component
What does the CRISPR-CAS9 complex do?
Cleaves invading DNA to prevent re-infection by viruses
How does CRISPR act as an adaptive immune regulator? (2)
- Invading DNA recognised and cut by Cas1-2 into fragments (protospacers)
- Protospacers integrated into CRISPR locus in bacterial genome
Describe what occurs upon viral reinfection with CRISPR. (4)
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
Describe the structure of the CRIPSR locus. (4)
5’ transactivating RNA
Cas operon encoding Cas components
Identical repeat arrays
Spacers of invading DNA
What is the guide RNA (gRNA)?
Complex formed between transactivating RNA (tracrRNA) and protospacer/CRISPR RNA (crRNA)
Enables selective binding of Cas9 to invading DNA sequences
Explain how protospacer adjacent motifs (PAM) allow Cas9-mediated DNA cleavage.
Cas9 will not cut invading DNA without PAM site irrespective of Cas/gRNA binding
Describe the location of protospacer adjacent motifs.
2-8 base pair sequence 3-4 base pairs downstream of cut site
Give the 2 steps of Cas9 function in initial infection.
Protospacer aquisition
Spacer integration
Give the 4 steps of Cas9 function in re-infection.
Transcription
Binding to Cas9
Sequence comparison
Cleavage
How is gRNA modified for biomedical studies?
Linking cRNA and tracrRNA by adding linker loop
= composite gRNA
Give 2 properties of correct gRNA design.
Contains protospacer sequence (target sequence) upstream of PAM site
Should be selective to a single genome locus to avoid off target effects
If DNA is damaged by ionising radiation, double stranded breaks are repaired of one of two which mechanisms?
Homology-directly repair (HDR)
Non-homologous end-joining (NDEJ)
Describe how non-homologous end-joining (NHEJ) enables error-prone DNA repair. (3)
Introduces insertions or deletions into DNA
Impacts gene function
Often results in premature stop codons
Describe how homology-directed repair (HDR) enables precise DNA repair.
DNA precisely repaired using sister chromatid during S phase of cell cycle
Key to CRISPR knock-in studies
Give the 3 steps to homology-directed repair after a double-strand break.
End resection
Strand invasion synthesis
Synthesis-dependent strand annealing
Describe CRISPR-mediated gene knockout via NHEJ. (5)
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
Describe CRISPR-mediated gene knock-in via HDR. (5)
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
Explain which receptor signalling is the main driver of prostate cancer. How is this useful in treatment?
Androgen receptors (AR)
Current treatment aims to inactivate AR by blocking ligand binding
What is castrate-resistant prostate cancer?
Relapsed, incurable prostate cancer
What 2 CRISPR-based studies are there for prostate cancer?
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
Describe what studies of androgen receptor variants have discovered so far. (2)
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
Why is it difficult to study AR-Vs in isolation?
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
How is CRISPR hoped to cure prostate cancer? (2)
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
Describe CWR22Rv1-AR-EK. (3)
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
Give 3 considerations of cell therapy.
Efficacy of delivery
Regulatory guidelines
Mosaicism
Specificity
Immunogenicity
Germline vs somatic
Describe ex-vivo delivery of CRISPR in the clinic. (4)
Remove cells from patient/donor
Edit genome
Screen/expand cell populations
Engraft cells back into patient
Describe in-vivo delivery of CRISPR in the clinic. (2)
Package CRISPR/Cas in a delivery vehicle
Deliver to patient
Describe the potential of CRISPR editing for HIV treatment.
Homozygous CCR5^32 donor gave stem cell transplant
Patient/recipient no longer required anti-viral therapy
Describe how modified CCR5 results in a resistance to HIV infection.
Modified CCR5 means virus cannot bind to receptor and infect cells
32bp deletion results in frameshift and unstable protein
Describe how successful CRISPR editing of CCR5 in vivo confers HIV-1 resistance.
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
Explain how CRISPR editing of CCR5 gene applies in humans.
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
