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Essential Biomed Research Skills > CRISPR: Lecture 12 > Flashcards

CRISPR: Lecture 12 Flashcards

1
Q

what does CRISPR and Cas stand for?

A

clustered regulatory interspaced short palindromic repeats
crispr associated proteins

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2
Q

what are the 3 components of the guide RNA protein complex?

A

Cas9
crRNA
tracrRNA

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3
Q

why is the tracrRNA in a loop/hairpin sructure?

A

it’s a palindromic sequence when its double stranded so when turned into single stranded rna it forms the loop.

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4
Q

what is the cut out part of the viral genetic material called in the CRISPR locus?

A

protospacer

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5
Q

what happens upon viral reinfection in bacteria (crispr) ?

A

Bacteria transcribe the crispr locus
crRNA and tracrRNA form the guide RNA which binds to cas9.
Cas9/RNA duplex anneals to complementary sequence which is next to PAM sequence and creates double stranded break in viral DNA

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6
Q

Structure of CRISPR locus left to right?

A

tracrRNA, cas operon, identical repeat array, protospacer

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7
Q

what does tracrRNA stand for?

A

transactivating RNA

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8
Q

what is the role of tracrRNA?

A

important role in pre-crRNA process and target recognition and cleavage

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9
Q

what does PAM sequence stand for and what it do?

A

protospacer adjacent motif. Located next to target sequence
cas protein cuts the target DNA if it recognises the specific PAM sequence.

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10
Q

How long is the PAM sequence and exactly where its located?

A

its 2-8 base pairs long
3-4 base pairs downstream of target DNA

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11
Q

How does Cas9/rna system void autoimmunity?

A

PAM sequence is not in crispr locus so it won’t cut its own DNA

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12
Q

How does Cas9/rna duplex distinguish self from non-self?

A

PAM sequence on the viral genetic material.

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13
Q

How does Crispr/cas9 get engineered for use?

A

Linker loop added between the tracrRNA and crRNA so it becomes a crRNA-tracrRNA chimera, composite guide RNA (gRNA)

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14
Q

How is the guide RNA formed?

A
  1. Crispr locus gets transcribed same time as tracrRNA
  2. repeat-spacer array is called pre-crRNA
  3. pre-crRNA gets chopped into sections called crRNA
  4. tracrRNA is complementary to the repeat section of the crRNA so it hybridises
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15
Q

How are cells manipulated to express cas9 and gRNA?

A
  1. Plasmids get transfected into the cell and get transcribed/translated to produce cas9 and gRNA
  2. Preformed RNA/protein complexes from purified cas9 and gRNA
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16
Q

crucial aspects of designing guide RNA?

A

gRNA should only be specific to one locus
gRNA should have protospacer sequence upstream of PAM site.

17
Q

What are the 2 types of DNA double stranded break repair in cell?

A

Homologous directed repair (HDR)
Non-homologous end-joining (NHEJ)

18
Q

How does NHEJ work?

A

After double stranded break, some of the DNA gets resected and then polymerase ligates the ends randomly. Creates indels resulting in stop codons and impact gene function.

19
Q

Which repair pathway is error prone and why? What is useful about this?

A

NHEJ because there’s no donor template to conserve the sequence so the repair produces indels with stop codons.
Useful for knockout studies

20
Q

How does homology directed repair work?

A

DNA break precisely repaired using sister chromatid as donor template in S phase.
5’ to 3’ degradation leaving 3’ end resection
Strand invasion and d-loop
synthesis of new strand and annealing of other strand.

21
Q

Which repair pathway is more precise?

A

HDR- homology directed repair

22
Q

What are the considerations when using HDR for knock in studies?

A

PAM sequence must be removed
point mutation/insert must have more than 60 bp on each side on the homology arms

23
Q

How does androgen receptor (AR) relate to prostate cancer?

A

AR signalling drives prostate cancer. AR is nuclear hormone receptor transcription factor that resides in cytoplasm. When bound to DHT (dihydrotestosterone) it becomes active and goes to nucleus to drive gene expression, hyperproliferation and anti-apoptotic.

24
Q

what are the knockout and knock in strategy outlines for crispr in prostate cancer?

A

Knockout- generate inducible cas9-expressing cell line to identify new targets
Knock in- create modified form of prostate cancer cell line to study aberrant forms of AR (AR-Vs)

25
Q

What is AR-Vs and how does it relate to prostate cancer?

A

short form of AR- made when cell adapts to full length AR targeting agents like enzalutamide.
AR-Vs lack exons 4-8 which code for LBD (ligand binding domain) the target of enzalutamide. So AR-Vs unaffected by these targeting agents and associated with cancer progression

26
Q

What’s the name of that AR targeting agent and how it works?

A

Enzalutamide. Binds to LBD- ligand binding domain and inactivates the AR

27
Q

How does the crispr knockout strategy work in detail for prostate cancer?

A

transfect prostate cancer cell lines w/ plasmid that codes for cas9 thats inducible by doxycycline.
knock out AR synthesis and looking for targets that reduce AR levels- see using immunofluorescence.

28
Q

How crispr knock in strategy for prostate cancer work in detail?

A

prostate cancer cell lines express long and short forms AR. Use crispr to knock in stop codon to exon 5 (LBD) of the long form AR. So cell now only express AR-Vs and their effects can be studied.

29
Q

what are the cell therapy considerations?

A

Efficacy of delivery
regulatory guidelines
mosaicism
germline vs somatic
immunogenicity
specificity- is it off target?

30
Q

what are the 2 forms of crispr delivery?

A

ex vivo and in vivo

31
Q

how does ex-vivo crispr delivery work?

A
  1. collect circulating cells from patient
  2. modify cells ex vivo
  3. expand cell populations
  4. engraft cells back into patient
32
Q

How does in-vivo crispr delivery work?

A

package crispr machinery into delivery vehicle
deliver to patient

33
Q

which is the receptor that gives natural HIV resistance?

A

CCR5delta32 homologous- deletion of this receptor. Heterozygous means delayed disease progression

Essential Biomed Research Skills (4 decks)

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