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CMB2000 > L13-Genome Editing > Flashcards

L13-Genome Editing Flashcards

1
Q

how many base pairs are in the human genome

A

3 billion base pairs

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2
Q

what is genome editing

A

a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases

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3
Q

what does genome editing lead to

A

i. enabling specific targeting of sequences within the genome without impacting the rest of the genome sequence

ii. Potential to cure genetic diseases in a patient specific manner

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4
Q

what is CRISPR?

A

Clustered Regulatory Interspaced Short Palindromic Repeats

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5
Q

what is Cas

A

CRISPR associated proteins

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6
Q

CRISPR-Cas9 complex is part of the Adaptive immune system of prokaryotes

A

True

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7
Q

CRISPR-Cas9 complex is Found in more than 40% of bacterial genomes

A

true

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8
Q

what are the components of CRISPR-Cas9 complex

A

1 Cas9 - Protein component

2 crRNA – RNA component

3 tracrRNA – RNA component

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9
Q

What does CRISPR-Cas9 complex do

A

cleaves invading DNA to prevent re-infection by viruses

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10
Q

how does CRISPR act as an ‘immune system’ in prokaryotes against invading DNA/RNA

A

1- Invading DNA recognised and cut by Cas1-Cas2 protein complexes into fragments termed protospacers

2- Protospacers integrated into CRISPR locus located in the bacterial genome

3- Upon viral reinfection, transcription of the protospacers to RNA is activated which bind to Cas9

4- Cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA

5- Cas9 cuts DNA strands creating a double strand break to prevent infection

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11
Q

what are the components of the CRISPR locus

A

Transactivating RNA

Operon of cas genes encoding Cas protein components (e.g. Cas1/Cas2/Cas9)

Identical repeat array

Spacer of invading DNA

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12
Q

what is the guide (gRNA)

A

The complex formed between the Transactivating RNA (tracrRNA) and the
Protospacer/CRISPR RNA (crRNA)

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13
Q

what does guide RNA (gRNA) do

A

enables selective binding of Cas9 to invading DNA sequences

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14
Q

what is the function of the cas operon

A

encodes Cas proteins required for DNA cleavage

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15
Q

what do Protospacer Adjacent Motifs (PAM) do

A

enable Cas9-mediated DNA cleavage

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16
Q

what are the features of PAM

A

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

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

PAM sequences are not present in the CRISPR locus

Prevents bacterial CRISPR locus being targeted by Cas proteins

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17
Q

what RNA is bound to cas9 and what RNA is does it contain?

A

gRNA contains crRNA and tracrRNA

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18
Q

what enables site-specific cleavage thro nuclease activity

A

Deposition of the Cas9/gRNA complex at a desired locus of the genome

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19
Q

what enables specific genomic edits to be introduced

A

The repair of the DNA break by endogenous DNA repair pathways

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20
Q

where should the proto-spacer sequence (target sequence) of the gRNA be located

A

upstream of the PAM site

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21
Q

why should the gRNAs be selective to a single genome locus

A

to avoid off target effects

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22
Q

what are the mechanisms of repairing DNA

A

Homology-directed repair (HDR)

Non-homologous end-joining (NHEJ)

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23
Q

what is produced when Cas9 cleaves DNA

A

a double strand break

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24
Q

how do HDR and NHEJ function

A

downstream

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25
Q

which DNA repair mechanism enables error-prone DNA repair

A

NHEJ

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26
Q

how is DNA repair by NHEJ error prone

A

introduces insertions or deletions (indels) into DNA

Impacts gene function

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27
Q

which DNA repair mechanism enables precise DNA repair

A

HDR

28
Q

when does precise DNA repair take place

A

S phase of the cell cycle

29
Q

what is used in precise DNA repair

A

sister chromatids

30
Q

what are the steps of CRISPR-mediated gene knock out via NHEJ

A

Target Cas9:gRNA complex to gene of interest

DSB introduced

Cell repairs the break via NHEJ
- Error prone

Indels introduced generating a frameshift
- Premature stop codons introduced

Normal gene product not expressed

31
Q

what does error prone DNA repair introduce

A

indels

32
Q

what do indels generate

A

a frameshift

33
Q

what are the steps of CRISPR-mediated gene knock-in via HDR

A

DSB introduced by Cas9:gRNA complex

Introduce a template that the cell will use to repair the DSB through HDR

HDR template requires >60 bp homology arms on either side of the point of mutation/insert

PAM sites are removed from HR template to prevent re-targeting of region

Inserts of several kilobases are possible

34
Q

what drives prostate cancer progression

A

androgen receptor signalling

35
Q

what does current treatment of prostate cancer aim to

A

inactivate AR by blocking ligand binding

36
Q

what is castrate resistant prostate cancer

A

an incurable form of prostate cancer

37
Q

what are the 2 CRISPR based studies regarding prostate cancer

A
  1. To generate a Cas9-expressing prostate cancer cell line to knock-out AR : Knock-out strategy
  2. To create a modified prostate cancer cell line to study function of aberrant forms of the androgen receptor: Knock-in strategy
38
Q

how is the AR gene knocked out to treat prostate cancer

A

a stop codon is introduced into the gene using specific Cas9 cleavage event through NHEJ error-prone DNA repair

39
Q

how are androgen receptor variants produced

A

by alternative splicing

40
Q

when are androgen receptor variants produced

A

when AR-targeting agents, such as enzalutamide, are present

41
Q

what is the target site for enzalutamide binding

A

the ligand-binding domain (LBD)

42
Q

what do AR-Vs lack

A

exons 4-8 which encodes the ligand-binding
domain (LBD)

43
Q

what does Loss of the AR LBD create

A

constitutively active
transcription factors that are refractory to enzalutamide

44
Q

what happens to the Expression of AR-Vs in advanced disease

A

its elevated

45
Q

what results in using CRISPR knock in strategy on AR gene

A

create an AR-V-only expressing cell line (blocking expression of full-length AR)

46
Q

what is full length AR encoded by

A

exons 1-8 of the AR gene

47
Q

what is gRNA designed for in AR gene knock in

A

exon 5 (LBD)

48
Q

what causes the point mutation in exon 5 of the AG gene

A

HDR

49
Q

what does the point mutation in exon 5 of the AR gene result in

A

a stop codon

50
Q

how does the introduced stop codon affect the AR in CRISPR knock in

A

stops the formation of FL-AR

51
Q

what is the name of the cell line that expresses both full-length AR and AR-Vs

A

CWR22Rv1

52
Q

what is the name of the cell line that expresses AR-Vs

A

CWR22Rv1-AR-EK

53
Q

what protein is involved in therapeutic vulnerability in prostate cancer cells expressing AR-Vs

A

PARP1/2

54
Q

what considerations should be in mind with regards to cell therapy

A

Efficacy of delivery

Regulatory guidelines

Mosaicism

Specificity - off target effects

Immunogenicity

Germline Vs Somatic

55
Q

what are the methods of delivering CRISPR in the clinic

A

in vivo

ex vivo

56
Q

how is CRISPR delivered in vivo

A
  1. Package CRISPR/Cas in a delivery vehicle
  2. Deliver to patient
57
Q

how is CRISPR delivered ex vivo

A
  1. Remove cells from the patient / donor
  2. Edit genome
  3. Screen / expand cell populations
  4. Engraft cells back into patient
58
Q

what is the story of the berlin patient

A

1995 diagnosed HIV positive, 2006 diagnosed with acute myeloid leukaemia

2007 and 2008 patient received hematopoietic stem cell transplant

Stem cell donor was homozygous CCR5Δ32

Patient no longer requires anti-viral therapy

59
Q

Individuals with homozygous CCR5Δ32 have a natural resistance to HIV infection

A

True

60
Q

what percentage of the white northern European population is homozygous CCR5Δ32

A

~1%

61
Q

Individuals heterozygous for the CCR5Δ32 allele have a delayed disease progression

A

true

62
Q

what percentage of the white northern European population is heterozygous for CCR5Δ32

A

~15%

63
Q

what does CCR5Δ32 result in

A

32 bp deletion of the coding sequence resulting in frameshift and unstable protein

64
Q

how does CRISPR editing of CCR5 confer HIV-1 resistance in vivo

A

Long term CCR5 disruption was observed

CCR5 disrupted HSCs were able to reconstitute a functional immune system

Viral titre reduction and increase in CD4+ T cells demonstrated HIV resistance

65
Q

why cant CRISPR editing of CCR5 confer HIV-1 resistance in humans

A

transplanted stem cells were unable to provide HIV resistance Due to low amounts of modified cells

CMB2000 (10 decks)

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