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Methods in molecular biology > Gene expression based on recombination > Flashcards

Gene expression based on recombination Flashcards

1
Q

Explain why homologous recombination is non-specific.

A

The carry-over of genetic material is not the function of homologous repair, it’s the consequence.

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2
Q

What’s site-specific recombination?

A

It’s when specific recombinases identify sequences are direct a recombination process.

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3
Q

There are two resolutions to the holliday junctions, what are they called?

A

Patch (Only one strand’s material is exchanged)

Exchange (both strands are exchanged.

Double-check if there are any other differences. I am not sure this is it.

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4
Q

Draw homologous repair.

A

Do it.

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5
Q

Explain the recBCD mechanism of action.

A

RecBCD mediates homologous recombination in bacteria.

The following occurs in both ways following a double-stranded break.

  1. recBCD recognizes free ends of DNA near dsDNA breaks.
  2. recBCD degrades (fragments) the strands (there’s nuclease activity).
  3. when a chi-sequence (there are ~1000 in e.coli) is recognize, the D-subunit in recBCD falls of.
  4. recBC prepares the DNA for recombination by stopping 3’ degradation, 5’ degradation keeps going. This generates a 3’ overhang.
  5. recA is recruited.
  6. recA assembles on the 3’ ssDNA and wraps around it.
  7. the ssDNA guides recA to homologous sequences in dsDNA.
  8. recA invades dsDNA, makes new heteroduplex DNA.

Comment: ssDNA is generally bound by single-stranded DNA binding protein (SBP), recA can bind to ssDNA due to it being recruited.

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6
Q

Other than homologous repair, what’s the function of recBCD?

A

DNA without chi-sequences are degraded. This makes e.coli hard to transform with linear DNA.

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7
Q

Explain how the lambda-red system works.

A

Lambda red relies on three genes: EXO, BET, GAM.

  1. GAM: inhibits recBCD (linear dsDNA can now be introduced)
  2. EXO: Attacks dsDNA (insert), creates 3’ overhangs.
  3. BETA: binds linear ssDNA and anneals it to the 3’ overhangs generated by exo.
  4. Homologous recombination for insertion of insert.
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8
Q

How long sequence homology must exist in the insert in order for homologous repair to occur?

A

40 nt.

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9
Q

Are there any problems with the lambda red system in e.coli? How do you tend to it?

A

Yes. GAM is toxic to e.coli, BETA and EXO reduce plasmid stability.

Make sure that the proteins’ promoter is stimulus-sensitive (so that you can regulate it).

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10
Q

Cosmids and BACs can carry huge plasmid, give an estimate.

A

45-300kb.

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11
Q

How can you increase the likelyhood of a plasmid’s content being inserted into a eukaryotic genome?

A

Stimulate a dsDNA break. Homologous recombination is not as active in eukaryotes as in bacteria, thus, stimulation is needed.

The lack of homologous recombination is an argument for why programmable nucleases are so important.

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12
Q

What’s TALENs endogenous function in xanthomonas?

A

The bacteria produce “TALeffectors” (TALEs). These are TFs that are secreted into plant cell nuclei and activate genes which facilitates a good environment for the bacteria.

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13
Q

What are the characteristics of TALEs?

A
  1. Tandem repeats of 34 aas.
  2. aa12 and aa13 contact a base in DNA.

You can engineer your own transcription factor.

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14
Q

How were TALEs converted into TALENs?

A

TALE + FokI (nuclease) = TALEN.

  1. Two TALENs are inserted, they bind 14-20 bp away from eachother.
  2. The 2 fokIs cut one strand each of dsDNA.
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15
Q

What’s the downside of TALENs compared to CRISPR/cas?

A

Lots of underlying genetic engineering is required.

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16
Q

What are the three DNA repair mechanisms that can repair a dsDNA break caused by programmable nucleases?

A

Homology-directed recpair

Homologous recombination

Non-homologous end-joining

17
Q

Explain the endogenous function of CRISPR/cas in e.coli.

A
  1. CRISPR/Cas is a form of adaptive immunity.
  2. Phage DNA fragments are stored in a clustered repeating interspaced short palindromic array.
  3. The array gets transcribed into crRNA.
  4. crRNA guids nucleases, like cas9 to complementary sequences, which are then cleaved.~

This needs to be fleshed out.

18
Q

Since crRNAs are surely complemntary to endogenous DNA, why doesn’t the nucleases cleave endogenous DNA?

A

The nucleases recognise PAM sequences which are not present in the bacterial genome.

Why doesn’t phages adapt? I THINK that the pam-sequences are very conserved. DIfferent cas-enzymes require different PAM sequneces.

19
Q

How many endonucleolytic domains does cas9 have?

A

2.

20
Q

Explain type 1 CRISPR systems (in endogenous circumstances)

A
  1. Phage infects bacteria w/ DNA.
  2. Cas enzymes acquire spacers (viral DNA) and installs it into the CRISPR array.
  3. CRISPR array is transcribed to pre-crRNA.
  4. pre-crRNA is cleaved with CRISPR-associated ribonucleases, generating small crRNAs.
  5. “The cascade complex” is guided to crRNA complementary sequence.
  6. the cascade complex recruits cas3 which cleaves the nucleic acid.
21
Q

Explain Type 2 CRISPR systems (in endogenous circumstances)

A
  1. Phage infects bacteria w/ DNA.
  2. Cas enzymes acquire spacers (viral DNA) and installs it into the CRISPR array.
  3. Trans-activating crRNA (tracrRNA) hybridises with the crRNA. WHERE DOES trcrRNA COME FROM??
  4. The dsDNA are cleaved into small crRNA-tracrRNA hybrids by RNase III.
  5. crRNA + trcrRNA guides cas9 to its target.
22
Q

Explain type 3 CRISPR systems (in endogenous circumstances).

A
  1. Phage infects bacteria w/ DNA.
  2. Cas enzymes acquire spacers (viral DNA) and installs it into the CRISPR array.
  3. pre-crRNA is cleaved by CRISPR-associated ribonucleases, generating crRNAs.
  4. crRNAs 3’ ends are shortened by RNAses.
  5. The mature crRNA are loaded into a Csm or Cmr (proteins) which degrade DNA and RNA respectively.
23
Q

Which CRISPR system (1-3) is common in the lab? How is it modified from its endogenous state?

A

Type 2.

Instead of having cas9 guided by a crRNA+trcrRNA hybrid, its guided by an artificial gRNA.

24
Q

Explain how a KO can be generated by CRISPR-cas9.

A
  1. Ensure that PAM sequences are present where cleavage is inteded. (Different cas9 variants have different PAM seqs).
  2. Design gRNA for the sequence.
  3. dsDNA break –> repair.
  4. Screen for mutants which have repaired the dsDNA break by NHEJ.
  5. Frameshift / point-mutations.
25
Q

Explain how you can insert genetic material into a genome with CRISPR/cas9.

A
  1. Ensure PAM seq is present.
  2. Design gRNA.
  3. Introduce repair template.
  4. dsDNA break –> Repair (three ways)
  5. Screen for mutants which have undergone homology-directed repair.
26
Q

How can CRISPR/cas9 be used for base editing?

A
  1. Inactive cas9 (can bind, cant cut)
  2. Fuse enzyme that causes nucleotide conversions (ex cytosine deaminase).
  3. cas9 guides editing enzyme to the right spot.
  4. If U is incorporated, cellular repair mechanisms correct it to T.
27
Q

Get a grasp of prime editing.

A

Do it.

28
Q

How can CRISPR/cas9 be used for gene supression (no KO)?

A
  1. Make cas9 catalytically dead.
  2. cas9 will bind to DNA, block RNApols from transcribing it.
29
Q

What’s site-specific recombination? Give an example of a method utilizing this.

A

It’s when a recombinase recognises accessory sites (upstream or downstream) of cleavage site, then recombines 2 copies of the recognized sequence.

Example: Cre-LoxP recombination.
1. LoxP-sites are recognised.
2. Depening on how the sequence is seated, the recombinase (cre) may invert, translocate or delete the sequence.

30
Q

How can Cre-LoxP be utilized when generating transgenic (KO) mice?

A
  1. Gene of interest is flanked by loxP sites and inserted into a mouse (homologous recombination?)
  2. LoxP mouse is mated with mouse carrying the cre recombinase.
  3. Some offspring will have Cre and LoxP. In these, inversion, translocation or deletion of the gene may happen.
31
Q

Is cre-loxP a disruptive method (when used in transgenic mice)?

A

No. When using it, the KO can be observed without disturbing any tissue.

Methods in molecular biology (23 decks)

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