CRISPR & Synthetic Biology

Question 1

What are the bacterial defense mechanisms against bacteriophage

Answer 1

1. mask outsite of cell so it can’t be recognized
2. block phage injection of genetic material
3. bacteria lyse itself to stop phage from replicating
4. Dam differentiates between host, methylated, and viral DNA and endonuclease cleaves non-meth DNA
5. CRISPR

Question 2

What are the steps of the CRISPR mechanism?

Answer 2

1. acquisition
2. expression
3. interference

Question 3

What is the acquisition steps of CRISPR?

Answer 3

• viral phage DNA injected
• bacteria takes small piece of viral DNA into CRISPR array and puts between palidromic repeats in cas locus

Question 4

What is the expression steps of CRISPR?

Answer 4

• second infection
• Cas proteins translated and pre-crRNA transcribed
• crRNA is discrete units containing hairpin loop and one viral DNA section

Question 5

what does pre-crRNA look like?

Answer 5

hairpin loops due to palidromic repeats + each viral information between

Question 6

What is the interference steps of CRISPR?

Answer 6

• crRNA is loaded onto Cas9 protein with tracrRNA
• tracrRNA bp with palidromic repeat of crRNA and viral segment bp with infector
• Cas9 acts on new viral DNA

Question 7

What is a protospacer?

Answer 7

DNA sequence in “foreign” genome that becomes the spacer in the bacterial genome that Cas9 binds

Question 8

what is PAM

What is it found by?

Answer 8

short conserved sequence next to protospacer (where Cas9 binds) in “foreign” genome

NGG for Cas9 system

found by Cas1 and Cas2 from Cas operon

Question 9

What is CASCADE?

Answer 9

Crisper ASsociated Complex for Antiviral DEfense
- multisubuntil protein/RNA complex for interference by processing pre-crRNA to crRNA

Question 10

What is the role of Cas9?

Answer 10

• holds tracrRNA-crRNA complex
• recognizes PAM sequence and binds to it using its PI domain
• checks complementarity between viral RNA and crRNA
• cleaves viral DNA

Question 11

How does Cas9 cleave viral DNA?

Answer 11

nuceases:
1. HNH: cuts 1/2 viral DNA strands
2. RuvC I, II, III: 3 domain protein; cuts other strand

HNH and RuvC can be mutated to only cut one strand or not cut at all –> “dead” Cas9 (dCas9)

dCas9 can be fused to transcription activators or repressors are used to regulate expression without DNA cleavage

Question 12

What does the gene locus look like for CRISPR?

Answer 12

tracrRNA gene + cas operon + CRISPR array

cas operon has Cas proteins

Question 13

Issue with “NGG” in Cas9 system?

Answer 13

• low specificity
• must be a structural requirement for Cas9 recognition of viralDNA
• likely that Cas9 proteins recognize viral DNA bc it has ends (non-circular)

bacterial genome is circular

Question 14

What is sgRNA? Purpose? Role?

Answer 14

single guide RNA
- ssRNA that is crRNA + tracrRNA
- fewer molecules = less risk of damage + dissociation
- cleaves PAM sequence with Cas9