W - BIOL 200 - 7, MOLECULAR BIOLOGY OF GENE TARGETING
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Q: How can you determine the function of a new gene?
A: Disrupt its expression and see what happens.
Q: What are 3 ways to inactivate a gene in yeast?
A: Replace normal gene, introduce repressor protein gene, degrade mRNA.
Q: Why use S. cerevisiae for gene targeting? (2 reasons)
A: Takes up foreign DNA easily; DNA recombines into genome.
Q: How do you test if a gene is essential for survival? (Steps)
A:
PCR amplify selectable marker (antibiotic resistance) with overlap.
Transform diploid yeast.
Recombine with target gene.
Q: What happens when you grow yeast with antibiotic?
A: Only recombined cells survive.
Q: What if gene is required for survival?
A: Half haploid cells die, half survive.
Q: How do you test what the gene does?
A: Shut off transcription.
Q: What does the GAL1 yeast promoter do?
A: ON with galactose, OFF without galactose.
Q: How does the GAL1 promoter test gene function?
A: Switch carbon source → see if protein stops → check effect.
Q: Best method to test how gene cyc promotes survival?
A: Place cyc under GAL1 promoter → grow in galactose → shift to glucose → observe.
Q: What are Zinc Finger Nucleases (ZFNs)?
A: DNA-binding zinc finger domain + DNA cleavage domain.
Q: How many zinc fingers can ZFNs have?
A: 3–9, each for ~3 bp.
Q: What is the cleavage domain in ZFNs?
A: FokI nuclease, works as dimer.
Q: What are TALENs?
A: TAL effector DNA-binding domain + DNA cleavage domain.
Q: What’s special about the TALE binding domain?
A: Conserved 33–34 aa repeat, 12th & 13th variable → binds specific DNA.
Q: How many nucleotides does each TALE bind?
A: One.
Q: What is the cleavage domain in TALENs?
A: FokI nuclease.
Q: What does CRISPR stand for?
A: Clustered Regularly InterSpaced Palindromic Repeats.
Q: What does sgRNA do?
A: Has target sequence + Cas9 interacting sequence.
Q: What is a PAM sequence?
A: Protospacer adjacent motif before target site.
Q: What does Cas9 do?
A: Binds target, makes double-strand break.
Q: How does CRISPR repair DNA? (2 ways)
A:
Non-Homologous End Joining (NHEJ) → deletion/insertion → gene inactivation.
Homology Directed Repair (HDR) → new DNA inserted.
Q: Natural CRISPR system: how do bacteria use it? (5 steps)
A:
Cleave phage DNA, insert in CRISPR array.
Transcribe mRNA with repeats + phage DNA.
Repeat binds tracrRNA → scaffold for Cas.
Re-infection: complex base pairs with phage.
Cas cleaves phage DNA.
Q: How is CRISPR used in eukaryotes?
A: Cas9 cuts at site chosen by guide RNA → inactivate or replace gene.
Q: What repair can cause gene loss?
A: NHEJ → deletion/frameshift.
Q: How can new genes be added?
A: HDR → donor DNA inserted at break site.
Q: The CRISPR/Cas9 system adapts what natural phenomenon?
A: Acquired immunity of prokaryotes against foreign DNA.
Q: What can CRISPR/Cas9 be used for? (4 examples)
A:
Repair mutated gene
Delete chromosome region
Disrupt/inactivate gene
Repress gene transcription
Note 
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