Lecture 12 - reverse genetics and genome editing

Question 1

What is reverse genetics?

Answer 1

Know the gene of interest first and then find a mutant, screen mutant populations for mutations in GOI using PPCR based approaches

Question 2

In reverse genetics how can you test DNA pools of insertion mutants?

Answer 2

-use different primer combinations so that if there is an insertion in the GOI PCR products will be detected

Question 3

What is the process of Targeting Induced local lesions in genomes? (TILLING)

Answer 3

1) Use DNA pools from mutants
2) PCR amplify different segments of GOI
3) During heating and cooling if a pool contains a single nucleotide repeat (SNP) a hetero duplex if formed
4) Heteroduplex is cleaved using CelI nuclease
5) Samples denatured and separated by electrophoresis
6) sequence individual sample to identify base change, some will lead to a change in protein sequence

Question 4

What are the functions of reverse genetics?

Answer 4

1-find the mutant in a gene when you know the GOI
2-gene silencing by RNAi
3-gene replacement by homolgous recombination

Question 5

What is the process of genome editing via plasmid integration in yeast?

Answer 5

1) linearised plasmid with two different direct repeats at the edge of the gap, homologous two two direct repeats in the chromosome
2) leads to dHJ formation and resolution as crossing over occurs
3) a pair of the left and right boardering direct repeats with chromosomal information in the centre, flanks the inverted plasmid section

Question 6

What is the process of genome editing via gene replacement in yeast?

Answer 6

1) linear plasmid with indirect repeats in the same direction at the end of the plasmid, homologous to regions in the chromosome boardering another piece of DNA
2) intervening segment from the chromosome replaced by linear plasmid

Question 7

What is the effectiveness of genome editing via plasmid integration and gene replacement?

Answer 7

• effective in yeast
• less efficient in animals
• nearly absent in plants

Question 8

How can the effectiveness of genome editing via plasmid integration and gene replacement be increased in animals and plants? What is needed?

Answer 8

• introducing dsbreaks into specific sites in the genome as dsbreaks need to be repaired and intrioduced DNA can serve as repair template
• a nuclease that cuts the genome at a specific location

Question 9

What are engineered zinc-finger nucleases?

Answer 9

-combinations of zinc finger domains fused to endonuclease Fok1

Question 10

What do an increased number of ZF domains lead to in engineered zink-finger nucleases?

Answer 10

higher specificty of the nuclease

Question 11

What is the process of ZFN-mediated gene targeting?

Answer 11

1) begin with you GOI and to one cell culture add ZFNs (a) and to a second cell culture add ZFNs and a mutant donor template (b)

Question 12

What is the process of ZFN-mediated gene targeting using a donor template?

Answer 12

1) begin with you GOI and to one cell culture add ZFNs and a mutant donor template
2 - Gene X is cut by ZFNs to induce dsbreaks
3) - staggered ends are then generated and 3’ overhangs created
4 - repaired using the mutant donor template through HR as 3’ ends invade donor template (homolgy directed repair)

Question 13

What is the process of ZFN-mediated gene targeting without using a donor template?

Answer 13

1) begin with you GOI and to one cell culture add ZFNs
2 - Gene X is cut at the desired location in the gene by ZFNs to generate dsbreaks
3 - this leads to NHEJ and a pool of mutants with an insertion or deletion (mGene X)

Question 14

How do bacteria manipulate plant gene expression?

Answer 14

By injection of TALEs through type III secretion apparatus through a translocon
Once inserted TALEs express target genes that promote infection

Question 15

What does TALE stand for?>

Answer 15

Transcription Activation Like Effector proteins

Question 16

From what plant can TALEs be isolated and what do they do?

Answer 16

TALEs isolated from the plant bacterium Xanthomonas

-induce the expression of specific genes in plants, reprogramming the plant cell for its own specific purpose

Question 17

What is the repeat structure of TALEs and how do they bind?

Answer 17

a conserved repeat structure and within each reapeat there are variable amino acids
-each repeat in the effector binds to a single base in the promotor of the plant target gene

Question 18

What is the specific structure of TALEs?

Answer 18

From right to left genomically
1-translocation - type III secretion signal
2-secretion - required for chaperone binding
3-Anchoring - repeat associates with thymidine
4-Repeat variable di-residues (RVDs) - amino acids 12 and 13 encode DNA specificity
5-DNA binding region - 33-35 amino acid repeats forming a superhelic
6-Effector binding element - docking site on DNA for sense strand
7-transport - up to three eukaryotic nuclear localisation signals
8-Activation - acidic transcriptional activation domain

Question 19

How can TALEs change their DNA binding specificity?

Answer 19

changing the amino acids in the variable region of repeats

Question 20

What is TALEN?

Answer 20

Tale nuclease

Question 21

What are the three nucleases used in genome editiing?

Answer 21

ZFN
TALEN
Meganuclease

Question 22

What is Meganuclease?

Answer 22

nuclease used in genome editing
derived from mobile introns
long recognition sites and therefore highly specific
-more difficult to modify specificity than with ZFN and TALEN

Question 23

How does repair take place in genome modification by sequence specific nucleases?

Answer 23

no repair template, repair takes place by NHEJ

Question 24

To what end can site-specific nucleases be used?

Answer 24

• to produce gene knockouts (insetions/deletions)

- to replace genes (gene therapy)

Question 25

What are four DNA cutting enzymes and what do they do?

Answer 25

Spo11: generates DSBs in meiosis, low specificity but with hotspots
Cre: Site specific recombinase in bacteriophage excision and integration
HO endonuclease: generates DSB in yeast to trigger mating type switch
RAG1/2: sequence-specific cleavage in Ig genes

Question 26

What are differences in the features of Site specific recombination and Homologous recombination?

Answer 26

Site specific recombination
-cut and hold
-no DNA synthesis
Homologous recombination
-bind and find
-DNA synthesis 
BOTH INVOLVE HOLLIDAY JUNCTIONS

Question 27

What are the different types of accidental damage and programmed damage and their repair?

Answer 27

Accidental damage: mutagens, UN, gamma rays
Repair: as accurate as possible but SOS as last resort

Programmed damage: meiosis, Ig gene variation
Repair:
Gene conversion in meiosis
Error prone repair in Ig genes

Question 28

What is the process of gene replacement in mice?

Answer 28

1) Introduce a DNA fragment containing alter gene into many cultured ES cells and let each cell proliferate to form a colony
more efficient
2) test for the rare colony in which the DNA fragment has replaced one copy of thenormal gene
3)Inject ES cells into early embryo
4) Hybrid early embryo is partly formed from ES cells
5) intoduce hybrid early embryo in pseudopregnant mouse
6)somatic cells of offspring are tested for presence of altered gene and selected mice bred to test for presence in germline cells

Question 29

How can the process of gene replacement be make more efficient in mice using nucleases?

Answer 29

when introduce a DNA fragment containing alter gene into many cultured ES cells, sequence specific nucleases make it more efficient, can also directly inject mRNA coding for TALEN into the embryos