Module 2 Section 6

Question 1

Site-specific recombination

Answer 1

• type of genetic recombination the only occurs at specific sequences
• involves movement of specialized nucleotide sequences (mobile genetic elements) between non-homologous sites
• procedure carried out by recombinases

Question 2

Effects of site-specific recombination

Answer 2

• can result in insertion, deletion, inversion, depending on arrangement of recombination sites
• can give rise to spontaneous mutations
• useful for inserting new/altered gene, deleting genes

Question 3

Cre-CoxP and Flp-FRT transgenes

Answer 3

• specific directional sequences placed into a genome (NOT naturally occuring)
• Cre and Flp are recombinase enzymes that recognize LoxP and FRT sites
• must be 2 or more LoxP or FRT sites in genome for system to work

Question 4

Cre-CoxP and Flp-FRT outcomes

Answer 4

1. if sites are inverted, the recombinase will invert the sequence, changing its orientation
2. if sites oriented in same direction, recombinase cleaves out intervening sequence, leave one reformed LoxP or FRT site

Question 5

Techniques to determine gene function

Answer 5

• delete gene to determine effect of its absence on cell function
  1. Zinc finger nucleases (ZFN)
  2. TALENs
  3. CRISPR/Cas systems

Question 6

About zinc finger nucleases (ZFN)

Answer 6

• uses zinc finger containing protein to recognize specific site and cleave it
• Zn finger domain characterized by single atom of Zn coordinated to 4 Cys residues or 2 His residues+2Cys residues
• consists of approx 30 AA, folds into Beta-alpha-beta structure
• AA residues within alpha structure on Zn finger contact 3 consecutive nucleotides in major groove of DNA
• Zn fingers can be stitched together in tandem, allows for specific recognition of DNA sequences 9-18 BP long

Question 7

About TALENs

Answer 7

• Similar to ZFNs
• DNA binding directed by transcription activator-like effector (TALE) domains
• recognize single BP
• can be linked together + fused to nonspecific nuclease domain (Fokl) to yield a TALE nuclease (TALEN)
• can be designed to inactivate genes/integrate foreign DNA sequences

Question 8

TALEN + ZFN limitations

Answer 8

• require modularity to recognize different DNA sequences
• need different designer enzyme for each site you want to recognize
• requires a lot of protein engineering

Question 9

About of CRISPR/Cas

Answer 9

• viral DNA seized by bacterial immune system incorporated into CRISPR regions to become spacers
• forms ‘guide RNAs’ that can for larger ‘seek and destroy’ structure with Cas9 the detects foreign DNA
• viral DNA fragments integrated into CRISPR locus
• Cas9 cleaves + inactivates viral DNA

Question 10

CRISPR/Cas9 for Genomic Engineering

Answer 10

• guide RNA (gRNA) and trans-activating CRISPR RNA (tracrRNA) fused into single guide RNA (sgRNA)
• guide sequence can be programmed in

Question 11

Cas9 nuclease domains in genomic engineering

Answer 11

• 2 domains, one cleaves DNA paired w sgRNA, other cleaves opposite DNA strand
• OPTION 1: inactivating one domain creates enzyme that produced just ssDNA break
• repaired using homology directed repair
• OPTION 2: if DSB introduced non-homologous end joining (NHEJ) simply joins loose ends
• error prone, usually disrupts gene