20: Genome defence

Question 1

Retroelements

Answer 1

Mobile genetic elements that are transcribed into RNA first before converted back to DNA and inserted elsewhere on the genome

Question 2

Transposon

Answer 2

A mobile genetic element that can excise itself and reintegrate elsewhere in the genome

Question 3

Interferon

Answer 3

Chemical messengers secreted by animal cells in response to viral infection that function to alert surrounding cells of the virus presence.

Question 4

RNA interference (RNAi)

Answer 4

Response that is triggered by the presence of dsRNA and results in the degradation of mRNA or other RNA transcripts homologous to the inducing dsRNA. => gene silencing

Not found in prokaryotes.

Dicer cleaves longer dsRNA molecules into fragments of 21-23bp (short interfering RNA, siRNA).

siRNA recognized by RISC complex => separating of siRNA. RISC tries to find complementary RNA seq.
Slicer (nuclease activity of RISC) degrades complementary RNA.

Allows investigation of gene function without the need to make mutants with altered or inactivated versions of a particular gene.

Does not depend on where the mRNA is produced.

Question 5

Micro RNA (miRNA)

Answer 5

Small regulatory RNA molecules of eukaryotic cells.
Regulates gene expression of the cell itself by blocking translation.
Bind to 3’-UTR of mRNA.

Can target mRNA encoding TFs. Involved in regulation of development.

Question 6

Dicer

Answer 6

A ribonuclease that cleaves dsRNA into 21-23 bp fragments.

Question 7

RNA-dependent RNA polymerase (RdRP)

Answer 7

Enzyme that uses an RNA template to produce a complementary RNA copy.

Not present in mammals. Have specific immune system => not so important

The dsRNA generated can act as a substrate for the Dicer enzyme to generate more (secondary) siRNA. => amplifies RNAi effect

Question 8

Experimental Induction of

RNA interference

Answer 8

1. A single DNA segment transcribed from a single promoter that generates a stem and loop structure.
2. A DNA segment flanked by two opposing promoters.
3. Two DNA segments, one being the inverse of the other and both having separate promoters.

Question 9

Contents of the CRISPR system

Answer 9

Memory bank:
Array of foreign DNA segments:
spacers/memories.
Alternating with identical repeated sequences (may be palindromic)

Mechanism for identification and destruction of incoming foreign DNA or RNA.

Question 10

CRISPR-Cas activity (Figure 20.9)

Answer 10

Spacer acquisition and adaptation:
- Sequence elements from invading 
  nucleic acids become 
  incorporated 
  at the 5' end of the CRISPR locus.
- Chosen invader sequences are 
  found close to the protospacer 
  adjacent motifs (PAMs).

Expression stage:
- CRISPR locus is transcribed and 
  processed into mature CRISPR- 
  targeting RNAs (crRNAs) with 8nt 
  repeat tag and a single spacer 
  unit.  

Interference stage:
- Mature crRNAs associate with Cas 
  proteins to promote the 
  degradation of complementary 
  nucleic acids. 
- Separate effector molecules for 
  DNA and RNA targets.

Question 11

Class I CRISPR systems

Answer 11

Use multiprotein crRNP effector complexes (cascades) to target recognition sequences and cleave target nucleic acid.

• Structurally complicated
• Majority of CRISPR systems

Cascade:
Recognizes one DNA strand through the complementary base pairing with guide crRNA and then Cas3 nuclease introduces a single strand break on the opposite strand.

Question 12

Class II CRISPR systems

Answer 12

• Only one effector protein needed
• CRISPR-Cas9
• Require PAM for interference
• Extensively used in genetic
  engineering.

tracrRNA = trans-activating CRISPR
RNA

The tracrRNA, RNase III, and Cas9
nuclease generate crRNA that combines with tracrRNA to generate a dual tracrRNA-crRNA for targeting.
DsDNA complementary to the guide RNA is cleaved by Cas9 nuclease, creating a blunt-end.

Question 13

Nonhomologous end joining (NHEJ)

Answer 13

Cellular repair mechanism that combines the ends of ds breaks into one seamless DNA molecule.

Can introduce insertion or deletion mutations.
=> unpredictable, error-prone

Question 14

Homology directed repair (HDR)

Answer 14

Cellular repair mechanism that uses homologous sequences to repair ds breaks.

DNA can be inserted into specific sites => more predictable, less off-target effects.

• Produces knockout or knockin
  mutations with more precision.

Question 15

Delivery of CRISPR systems into plants

Answer 15

Particle bombardment:
- Coating metals with foreign DNA
and firing them at high rate
towards target cells.

Agrobacterium-Mediated Delivery:
- DNA containing cas9 and sgRNA 
  cassettes are transformed into 
  Agrobacterium
- Infection of plant cells => delivery 
  of DNA.

Require introduction of a foreign nuclease

Question 16

Gene drives

Answer 16

Genes that are inherited at much higher rates than dictated by Mendelian inheritance.
Can spread quickly through a population.

Without a gene drive and using Mendelian inheritance, only 50% of progeny would inherit a modified copy of a gene.

Is advantageous to integrate CRISPR systems into gene drives.

Question 17

Zinc finger nuclease (ZFN) genome editing

Answer 17

Artificial genome editing complex consisting of several zinc fingers comprising a DNA binding domain that are fused to FokI nuclease domain.

Usually present in TFs

Don’t require PAM sequences => theoretically unlimited in potentially targets.
Fewer off-target effects than CRISPR