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PMCOL 200 > CRISPR > Flashcards

CRISPR Flashcards

1
Q

What is genome-editing?

A

Cutting genomes using ‘DNA scissors’; CRISPR/Cas9; can be used to remove/add DNA to change the function.

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2
Q

What are genomics?

A

The study of the entirety of an organism’s genes.

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3
Q

What is Genome-wide association study (GWAS)?

A

A tool used in genetics research to associate specific genetic variations with particular diseases.

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4
Q

What are functional genomics?

A

A study that describes gene functions and interactions.

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5
Q

What are ZFNs?

A

Directed evolution.

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6
Q

What are TALENs?

A

Clone new RVDs.

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7
Q

When did CRISPR-Cas9 explode into popularity?

A

2012 onset.

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8
Q

Who discovered the CRISPR sequence?

A

Yoshizumi Ishino

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9
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Palindromic Repeats

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10
Q

When did the CRISPR win a Nobel prize for genome editing?

A

2020

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11
Q

What is Cas9?

A

Protein part of the prokaryotic immune system that defends against viruses,.

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12
Q

What recruits Cas9 to DNA target sites?

A

tracrRNA:crRNA duplex.

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13
Q

What are the key elements of the CRISPR/Cas9 components?

A
  1. Cas9 DNA endonuclease.
  2. crRNA, which contains 20bp sequence complementary to target.
  3. Trans-activating crRNA (tracrRNA) which acts as a bridge between crRNA and Cas9 enzyme.
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14
Q

What is PAM?

A

Protospacer Adjacent motif (NGG)

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15
Q

What is the mechanism of CRISPR/Cas9 DNA cleavage?

A
  1. Dna endonuclease targets a DNA sequence via (sgRNA) single guide RNA sequence upstream of the PAM. This results in a 3bp ds break upstream of the NGG.
  2. Resulting dsbs are repaired by either NHEJ or homology-directed repair (HDR).
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16
Q

What are the off-target effects of CRISPR?

A
  1. DNA bulge caused by 1-bp insertion.

2. RNA bulge caused by 1-bp deletion.

17
Q

What is the target sequence?

A

The 20 bp upstream the PAM.

18
Q

What is crRNA?

A

Crisper RNA; 17-20 nucleotide sequence to target DNA.

19
Q

Where does Cas9 cleave in the target DNA?

A

3 bases upstream the PAM.

20
Q

What is absolutely required for Cas9 cleavage?

A

The presence of a PAM.

21
Q

How may CRISPR be delivered? (form)

A
  1. DNA/RNA molecule encoding the Cas9 gene.

2. Functional ribunucleoprotein.

22
Q

How may CRISPR cross the membrane?

A
  1. Microinjection
  2. Electroporatin
  3. Chemical methods.
23
Q

What is the function of Cas nickase?

A

Makes edits in CRISPR to replace target DNA segments (specifed target base) SINGLE STRANDED CUT

24
Q

What is the function of gRNA?

A

Targets Cas to a specific locus.

25
Q

What are the components of the Base Editing complex?

A

Cas nickase, gRNA, target base.

26
Q

What components are involved in prime-editing?

A

Engineered reverse transcriptase fused to Cas9 nickase

Prime-editing RNA guide (pegRNA)

27
Q

What is the function of CRISPRa?

A

Used as transcription activators to increase gene expression.

28
Q

What is the function of CRISPRi?

A

Used as a transcription suppressor to decrease gene expression.

29
Q

What is the complex used in epigenome editing?

A

dCas9 with core catalytic domain of p300.

30
Q

What is the mechanism of epigenome editing?

A

Acetylation of target gene synergizes with activation of transcription factors and RNA Pol, resulting in upregulation of transcription.

31
Q

How is CRISPR used in screening?

A
  1. Screens for novel genes that regulate known phenotypes.
  2. Screen for drug resistance.
  3. screen for tumor metastasis and cell viability.
32
Q

When is in vivo CRISPR editing useful?

A

Diseases of the eye and ear.

33
Q

When is ex vivo CRISPR editing useful?

A

Blood diseases (sickle cell, hemophilia)

34
Q

When is somatic cell CRISPR useful?

A

Any cell not included in reproduction.

35
Q

When is germline cell CRISPR useful?

A

Cells in reproductive line.

PMCOL 200 (19 decks)

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