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Pre-Vet 2 > 11. Gene Editing > Flashcards

11. Gene Editing Flashcards

1
Q

What is gene editing

A

changing the nucleotide sequence at a specific chromosomal locus to any desired sequence

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2
Q

DNA endonuclease

A

used to cut the DNA at specific target locations within the genome

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3
Q

Results of DNA endonuclease

A

double-stranded breaks in the DNA - allows for genes to be modified

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4
Q

What happens when DNA is not repaired after DSB?

A

chromosomal instability and incomplete replication in the genome - cell death, cancer, other mutations

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5
Q

Nonhomologous end joining (NHEJ)

A
  • allows for gene knockouts/downs
    1. Proteins recognize double stranded breaks
    2. protein complex attaches to the end of the broken DNA
    3. The ends are trimmed to remove nucleotides until a blunt end is achieved
    4. DNA ligase glues the blunt ends back together.
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6
Q

Homology directed repair (HDR)

A
  • allows for gene insertion/modifications
    1. A DSB occurs
    2. Nucleases trim off a portion of the broken strand RAD15 binds the undamaged chromatid
    3. Strand invasion occurs resulting in a displacement loop
    4. DNA replication within the D loop synthesises new DNA using the sister chromatid as a template
    5. DNA strands are trimmed to blunt ends and are ligated to finish repair
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7
Q

Compare NHEJ and HDR:
1. Nucleotide trimming?
2. Prone to error?
3. Uses a DNA template?
4. When does it occur?
5. Result?

A
  1. Yes and yes
  2. Yes and no
  3. no and yes
  4. before DNA rep and after DNA rep
  5. Gene knockdowns/out and gene knock ins
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8
Q

What do gene editing tools do?

A
  • exploit the cells natural DNA repair mechanisms
  • translationally fused to a sequence-specific DNA binding domain
  • Incorporated into a complex with an RNA molecule
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9
Q

Compare ZFNs and TALENs
1. Origins
2. Structure
3. Target Organisms
4. Endonuclease
5. Cleavage method
6. Drawback

A
  1. african clawed frog and xanthomonas
  2. DNA-binding loop with zinc finger-like structures and Tandem array of DNA binding repeats
  3. Somatic and pluripotent stem cells in humans and yeasts, fruitflies, roundworms
  4. foki and fokie
  5. dimerise and dimerise
  6. costly, difficult to produce, off target cleavage and costly/off target cleavage
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10
Q

CRISPR/Cas9

A

Clustered regularly interspersed short palindromic repeats
- found DNA nucleases encoded in the genome (CRISPR-associated cas genes)
- natural defense mechanism in prokaryotes

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11
Q

CRISP/Cas 9 is a natural defense mechanism in prokaryotes, how?

A
  1. sequences are transcribed into non-coding RNA
  2. tracer RNA gene is also transcribed into tracrRNA and cas9 is produced
  3. processed into crRNAs by cas-encoded RNases
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12
Q

tracrRNA and crRNA form a complex with Cas endonucleases to cleave foreign DNA, how?

A
  1. tracrRNA binds to the Cas endonuclease
  2. crRNA binds to tracrRNA via complementary base pairing
  3. the tracrRNA-crRNA-Cas complex cleaves the invading DNA into a DBS, deactivating the invader
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13
Q

What can the bacteria store and for what?

A

It can store the cleaved DNA in its genome for future invader recognition or inheritance
- can incorporate the cleaved foreign DNA into its CRISPR region

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14
Q

What have scientist changed with CRISPR?

A
  • re-engineered CRISPR to target any sequence of interest
    1. crRNA and tracrRNA are fused together into single guide RNA
    2. scientist can then insert their own target DNA into the sgRNA gene - creating a unique crRNA
    3. Cas9 is used as the endonuclease
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15
Q

One Cas-9 make the cut, endogenous DNA repair finishes CRISPR/Cas9 gene editing

A
  1. NHEJ repair of double stranded breaks results in deletion of one or more nucleotides
  2. NHEJ can sometimes introduce insertions of DNA derived from elswehere in the genome
  3. HDR uses supplied DNA fragments as the template for DNA syntehsis
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16
Q

HNH domain

A

cleaved the strand complementary to the crRNA

17
Q

RuvC domain

A

cleaves the opposite strand of DNA

18
Q

PAM

A

protospacer adjacent motif, destabilises the adjacent sequence for crRNA complementary paring

19
Q

Agrobacterium mediated transformation

A
  1. gene of interest is transformed in plasmid
  2. plasmid inserted in agrobacterium
  3. plant tissue is cocultured with agrobacterium
    4, plant is grown to observe change in phenotype
20
Q

Agrobacterium infiltration

A
  1. gene of interest is transformed in plasmid
  2. plasmid inserted in agrobacterium
  3. plant tissue is injected with agrobacterium
    4, plant is grown to observe change in phenotype
21
Q

Agrobacterium bombardment

A
  1. gene of interest is transformed in plasmid
  2. plasmid coated onto gold particles
  3. plant tissue is bombarded with particles
    4, plant is grown to observe change in phenotype
22
Q

What does agrobacterium do

A
  • it can alter the host nuclear genome
  • plant pathogen can infect the host and cause uncontrollable cell division
23
Q

What do agrobacterium contain

A
  • a plasmid called tumor inducing plasmid (Ti plasmid)
24
Q

Transfer dna

A

A portion of the plasmid can transfer to the plant and recombine into the host nuclear genome

25
Q

In agrobacterium medited transformation what is transcribed/translated

A

T-DNA is transcribed and randomly inserted into the nuclear genome
virulence proteins are transcribed and translated
conjugative transfer proteins are “

26
Q

How can scientists use agrobacterium to transfer any gene to plants?

A
  1. the Ti plasmid is disarmed by removing the opine- and tumor- inducing genes from T-DNA
  2. The gene of interest is inserted between the left and right r-dna BORDERS INTO A TRANSFORMATION Vector
  3. the 2 plasmids are reinserted into agrobacterium
27
Q

genes on the disarmed plasmid are turned into

A

virulence and conjugative proteins

28
Q

What is CRISPR/Cas9 mostly used for and where are the edits

A
  • plant biology research
  • economically important plants
  • most edits are via NHEJ repair to promote gene knockouts/downs to determine gene function in model plants
29
Q

Grey area in CRISPR

A

GMOS - orgniasms that have had their DNA changed by artificial means
- some countries allow GMOs as long as the genetic change could have been done by selective breeding

30
Q
A

Pre-Vet 2 (13 decks)

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