Test 3 CRISPR/Cas

Question 1

Describe how the CRISPR/Cas system works in bacteria?

Answer 1

• Foreign DNA acquisition
  
  • Viral DNA is cleaved and added to CRISPR array.
  • Transcription of locus
• CRISPR RNA processing
  
  • CRISPR RNA is cleaved into gRNAs by nuclease.
  • The nuclease must be complexed with tracrRNA.
• RNA-guided targeting of viral element.
• Inactive Cas9 nuclease binds to gRNA and tracrRNA and becomes activated.
  
  • Recognizes a complementary target sequence and cleaves it.
• RESULT: Recombination or Deletion.

Question 2

Why is Cas9 inactive until it binds to gRNA and tracrRNA complex?

Answer 2

If it was activated it could spontaneously react to any DNA nearby.

Question 3

Two types of nuclease-induced genome editing.

Answer 3

• Homology directed repair:
  
  • Harder to do but fewer errors
• Non-homologous end joining
  
  • Extremely error prone.

Question 4

Describe the CRISPR/Cas system?

Answer 4

• CRISPR array:
  
  • Protospacers and short repeats
• Pre-crRNA is then joined with RNase 3, tracrRNA, and Cas9
• The complex cleaves the crRNAs
• The complex then looks for a target sequence (protospacer) which must be adjacent to a PAM (Protospacer adjacent motif).
• The complex then binds downstream (3’ ) of PAM sequence
• Cleaves both strands upstream of PAM
• Then the complex unbinds.

Question 5

Are tracrRNA and gRNA comined into one in the Lab?

Answer 5

YEA

Question 6

2 components needed for Lab based CRISPR/Cas9

Answer 6

Guide RNA (gRNA) - contains both tracrRNA and gRNA

Cas9 nuclease

Question 7

Explain the PAM sequence

Answer 7

• Specific for each bacterial species
• Found directly downstream of the target.
• Required for Cas9 to work.
  
  • Cas9 partially unwinds the the DNA.
• Thought to be present in every gene.

Question 8

Requirements of CRISPR/Cas in a lab

Answer 8

• The sequence is unique compared to the rest of the genome.
• The target is present immediately upstream of a PAM
  
  • PAM sequence is NGG.

Question 9

What does homology directed repair require?

Answer 9

A template

Question 10

Explain Cas9 nuclease

Answer 10

• Two endonuclease activities are present in a complex.
  
  • This means you can inhibit one and then selectively nick a single strand.
    
    • Using two different Cas9 complexes each with one domain inhibited. You can make a large change to the genome by cutting only one strand a distance apart. Then using a template (homologous directed repair) to splice it in.

Question 11

Doubly inhibited Cas9 can be used for what?

Answer 11

To hold a transcriptional activator or repressor to activate or repress a target gene.

Hold GFP to ID a specific segment of the chromosome.

Question 12

How can catalytically dead Cas9 be used?

Answer 12

• Neutered Cas9 can be used to activate or inactivate transcription of histones
  
  • By binding a methylase to the complex DNA can be methylated using Cas9.
    
    • Methylation decreases gene expression
  • Demethylase can also be used.
    
    • Demethyation increases gene expression.

Question 13

Limits of CRISPR/Cas in area of effect?

Answer 13

• Germline cells can be modded to effect whole animal.
• Somatic cells can be modded to effeect specific cells or tissues.

Question 14

Application of CRISPR to treat cancer

Answer 14

• IF an activated protein is causing cancer you could code a stop codon before the protein, and add the DNA back into the patient.
  
  • This would knock out the mutated protein.

Question 15

Uses of CRISPR in the clinic

Answer 15

• Precision therapy
  
  • Must ensure gRNA cant also bind elsewhere or there will be unintended side effects.
• Disease modeling
• Drug screening