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PMCOL 200 > 34 Gene Editing > Flashcards

34 Gene Editing Flashcards

1
Q

What is genomics?

A

Branch of biology dealing with aspects of the genome (entire collection of genetic info in an organism)

Mainly descriptive

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2
Q

What is GWAS?

A

Genome-wide association studies

  • can be used to identify disease-associated genes
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3
Q

What is predictive modelling?

A

Predict antibiotic resistance, susceptibility to deisease, rxns to drugs (pharmacogenomics)

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4
Q

What are zinc finger domains?

A

Ubiquitous small protein motifs which bind to a specific DNA triplet sequence (eg 5’GAA’3)

  • typically part of transcription factors
  • Each zinc finger domain binds to 3 bases of DNA
  • can be linked linearly to target larger sequences
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5
Q

What is a Zing finger nuclease? (ZFN)

A
  • Two zinc finger arrays linked to subunits of the heterodimeric FokI endonuclease
    • enzyme is split into 2 different pieces; they must come together to cut DNA
    • Individual FokI units are inactive but when heterodimer is formed they cleave DNA
    • The ZFNs bind to the left and right sequences surrounding the spacer where the cut occurs
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6
Q

What are TALEs?

A

Transcription Activator Like Effector

  • Produced by plant pathogen
  • Each TALE domain binds 1bp of DNA
    • can be linked to form arrays that target longer sequences
  • Within each repeat domain, 2 aa at positions 12 and 13 dictate which specific base is bound
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7
Q

What are TALE nucleases? (TALEN)

A

TALEN = two TALE arrays linked to subunits of the heterodimeric FokI endonuclease

  • no protein engineering required since each base can be targeted by a particular domain and domains can be independently linked together to form arrays
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8
Q

What are two main mechanisms in which DNA double-strand breaks can be repaired?

A
  1. Non-homologous end joining (NHEJ)
    • inactivation/knockout
  2. Homologous recombination (HR)
    • knock-in or gene editing by supplying repair DNA
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9
Q

How does NHEJ repair DNA ds breaks?

A

Non-homologous end-joining

  • induces insertions or deletions that inactivate a gene
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10
Q

How does Homologous recombination repair DNA ds breaks?

A

HDR repairs the DNA break using information contained in a repair template

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11
Q

What are the four steps of CRISPR/Cas9

A
  1. Invasion
  2. Adaptation
  3. Production
  4. Targeting
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12
Q

What are the three components of the CRISPR/Cas9 mechanism?

A
  1. Cas9 (endonuclease)
  2. crRNA - contains a 20bp sequence used to target
  3. Trans-activating crRNA (tracrRNA)
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13
Q

Cas9 displays inherent specificity for a ______ sequence

A

Cas9 displays inherent specificity for a PAM sequence

PAM = Protospacer Adjacent Motif

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14
Q

What is the sequence of events for the mechanism of CRISPR/Cas9 DNA cleavage?

A
  1. Cas9 binds PAM (5’NGG-3’) N=A,G,C,T
  2. After PAM binding, cas9 tries to pair guide RNA with DNA
  3. Cas9 contains two nuclease domains and each of these cuts one strand of DNA = ds break repaired via NHEJ or HDR
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15
Q

How are gene editing tools delivered to cells? (3)

A
  1. Viruses
  2. Physical disruption of the membrane
    • microinjection
    • electroporation
  3. chemical coating/tethering methods
    • Cas9-CPP/NLS
    • Lipid nanoparticle (LNP)
    • AuNP (gold nanoparticle)
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16
Q

What is the difference between Somatic cells and Germline cells?

A
  • Somatic cell:
    • Any cell in a living organisms other than reproductive cell
    • could involve many tissues
  • Germline cell:
    • involved in the reproductive process that are passed on to progeny (eggs, sperm)
17
Q

What is the difference between Ex-vivo therapy and In vivo therapy?

A
  • Ex-vivo
    • performed outside of the body
      • useful for blood diseases
  • In vivo
    • directly treats
    • involve injection of gene editing agents directly into the body
18
Q

Somatic therapy vs Germ Line therapy?

A
  • Somatic Therapy:
    • Gene inserted into body cells
    • Modification restricted to individual patient
    • Considered safe and ethical
  • Germ Line Therapy:
    • Gene inserted into germ line cells (become gametes)
    • Modification will be inherited by offspring
    • Not considered safe or ethically sound

PMCOL 200 (30 decks)

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