Lecture 6 - horizontal gene transfer (post-midterm)

Question 1

Purpose of DNA restriction/modification systems

Answer 1

Helps protect cell from deleterious foreign DNA (ex: phage DNA)

Question 2

Mechanism of DNA restriction/modification systems

Answer 2

• modification enzymes in cells methylate host DNA at specific sequences
• restriction endonucleases cleave unmodified DNA
• functions as a bacterial “immune system” to destroy any non-self DNA

Question 3

What do restriction enzymes cleave?

Answer 3

Most of the time, dsDNA. Only cuts at specific sites

Question 4

Downside of restriction enzymes

Answer 4

Recognizes short sequences that can be present in high quantities in the genome. Not very site-specific

Question 5

Where was CRISPR first discovered?

Answer 5

E. coli K12 (note: CRISPR is not active in K12)

Question 6

Components of the CRISPR system

Answer 6

1. cas genes: encode proteins that facilitate the CRISPR system (part of the palindromic repeat)
2. spacers that have a variable sequence (from phages); size varies

Question 7

How are sequences for integration chosen?

Answer 7

Sequences are always near PAM motifs in phage DNA.
- PAM motifs (NGG) are only seen in phage DNA, so these sequences won’t be cut when in the bacterial DNA
- bacteria doesn’t incorporate the PAM sequence

Question 8

Difference between sequences for restriction enzymes and CRISPR

Answer 8

CRISPR sequences are longer (usually 26-72 base pairs) –> more specific

Question 9

Components of the CRISPR-Cas complex

Answer 9

1. Cas complex (proteins encoded by the repeat)
2. crRNA: phage DNA copy
3. slicer (another cas protein for DNA cleavage)

Question 10

What is special about CRISPR/Cas9?

Answer 10

Cas9 is a single cas protein that performs all of the jobs of the cas complex

Question 11

crRNA+tracrRNA vs gRNA

Answer 11

• crRNA+tracrRNA: tracrRNA is base paired to a portion of the crRA
• gRNA: tracrRNA is fused with the crRNA

Question 12

Function of CRISPR-Cas in genome editing

Answer 12

• double stranded break from CRISPR is resolved thru NHEJ –> deletions/insertions that inactivate the gene
• double stranded break from CRISPR is resolved through homologous recombination –> specific alteration of a gene