Lecture 12 - reverse genetics and genome editing Flashcards
What is reverse genetics?
Know the gene of interest first and then find a mutant, screen mutant populations for mutations in GOI using PPCR based approaches
In reverse genetics how can you test DNA pools of insertion mutants?
-use different primer combinations so that if there is an insertion in the GOI PCR products will be detected
What is the process of Targeting Induced local lesions in genomes? (TILLING)
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
What are the functions of reverse genetics?
1-find the mutant in a gene when you know the GOI
2-gene silencing by RNAi
3-gene replacement by homolgous recombination
What is the process of genome editing via plasmid integration in yeast?
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
What is the process of genome editing via gene replacement in yeast?
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
What is the effectiveness of genome editing via plasmid integration and gene replacement?
- effective in yeast
- less efficient in animals
- nearly absent in plants
How can the effectiveness of genome editing via plasmid integration and gene replacement be increased in animals and plants? What is needed?
- 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
What are engineered zinc-finger nucleases?
-combinations of zinc finger domains fused to endonuclease Fok1
What do an increased number of ZF domains lead to in engineered zink-finger nucleases?
higher specificty of the nuclease
What is the process of ZFN-mediated gene targeting?
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)
What is the process of ZFN-mediated gene targeting using a donor template?
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)
What is the process of ZFN-mediated gene targeting without using a donor template?
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)
How do bacteria manipulate plant gene expression?
By injection of TALEs through type III secretion apparatus through a translocon
Once inserted TALEs express target genes that promote infection
What does TALE stand for?>
Transcription Activation Like Effector proteins
From what plant can TALEs be isolated and what do they do?
TALEs isolated from the plant bacterium Xanthomonas
-induce the expression of specific genes in plants, reprogramming the plant cell for its own specific purpose
What is the repeat structure of TALEs and how do they bind?
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
What is the specific structure of TALEs?
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
How can TALEs change their DNA binding specificity?
changing the amino acids in the variable region of repeats
What is TALEN?
Tale nuclease
What are the three nucleases used in genome editiing?
ZFN
TALEN
Meganuclease
What is Meganuclease?
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
How does repair take place in genome modification by sequence specific nucleases?
no repair template, repair takes place by NHEJ
To what end can site-specific nucleases be used?
- to produce gene knockouts (insetions/deletions)
- to replace genes (gene therapy)
What are four DNA cutting enzymes and what do they do?
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
What are differences in the features of Site specific recombination and Homologous recombination?
Site specific recombination -cut and hold -no DNA synthesis Homologous recombination -bind and find -DNA synthesis BOTH INVOLVE HOLLIDAY JUNCTIONS
What are the different types of accidental damage and programmed damage and their repair?
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
What is the process of gene replacement in mice?
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
How can the process of gene replacement be make more efficient in mice using nucleases?
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
