Regulatory RNA's and Crispr

Question 1

Cis and trans sRNA

Answer 1

• can be adjacent to the gene they control (often in plasmids)-cis
• can be separate from the genes they regulate- trans

Question 2

sRNA Regulation

Answer 2

• usually interfere with translation
• cis sRNAs have extensive homology, very specific
• trans sRNA have limited homology with their target, require the assistance of Hfq
• Hfq is protein that stabilizes RNA-RNA hybrids

Question 3

Riboswitches

Answer 3

-transcripts where the RNA is regulatory and controls gene expression

Question 4

What mechanisms do bacteria use to destroy incoming foreign DNA?

Answer 4

• restriction modification system
• block phage attachment
• block phage infection
• CRISPR

Question 5

CRISPR

Answer 5

• Clustered
• Regularly
• Interspaced
• Short
• Palindromic
• Repeats

Question 6

CRISPR History

Answer 6

• identified as a bizarre DNA sequence
• later was found to be associated with a few adjacent genes: CRISPR associated genes (CAS)
• we now know that CAS genes are enzymes that process the CRISPR locus

Question 7

CRISPR Locus

Answer 7

• DNA in between the repeats has homology to phage and plasmid DNA
• idea arose that these may be involved in protecting against invading DNA
• experimental evidence followed showing that the CRISPR locus expand after encounters with phage, then these bacteria were resistant to subsequent attacks by those phage

Question 8

Stages of CRISPR

Answer 8

1. Adaptation/Immunization: new spacer is acquired and put into CRISPR locus
2. crBiogenesis: the CRISPR locus is transcribed and the Cas proteins process the crRNAs
3. Interference: the target crRNAs guide an endonuclease to a target DNA that is then cleaved

Question 9

Targeting of foreign DNA: the role of PAMs

Answer 9

• in the CRISPR locus the photospacer is the target DNA that will be acquired from the phage or plasmid and incorporated as a spacer
• the photospacer adjacent motif (PAM) is a sequence next to the target sequence
• the PAM is not taken up and processed into the spacer
• the PAM sequence allows crRNAs to recognize and destroy them
• lack of PAM sequence protects against self cleavage

Question 10

Discrimination of self and non-self DNA

Answer 10

• the spacer sequence of the crane have complementarity with the CRISPR locus from which they are transcribed-have potential to target CRISPR DNA
• autoimmune response prevented by differential base pairing of spacer/target 5’flanking sequences
• nterference requires crRNA-target mismatches outside the spacer sequence
• occurs in S epidermidis, other organisms may use variations on this theme (e.g. 3’ flanking sequence)

Question 11

CRISPR Types

Answer 11

• 3 types of CRISPR systems
• classified by the way the crRNAs are processed
• most commonly discussed is Type II that uses Cas9 endonuclease

Question 12

How does CRISPR work?

Answer 12

1. CRISPR array is transcribed
2. Cas gene products process array
3. crRNA has a piece that is bound by the endonuclease (cas9)
4. crRNA-cas9 complex can recognize specific DNA sequences

Question 13

CRISPR in genome editing

Answer 13

• the ability to bring an endonuclease to any specific DNA site open up the possibility for genome editing
• CRISPR now used for genetic manipulation in eukaryotic cells