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what are the general aims of someone wanting to edit a genome?

people want to be able to target a a specific area of the genome and induce double stranded breaks with no off target effects with very efficient cut rates


what are the two response to double strand breaks?

non-homologue end going or homologous recombination


what normally happens when double strand breaks are repaired by no homologues end joining?

mistakes are made by the repair machinery and point mutations, deletions, insertions which causee frameshift and maybe truncated protein (KO)


what are the three main genome editing tools used?

zinc finger nucleases, CRISPR/Cas9, TALENS


what is the general structure of a zinc finger motif?

a 2 strand beta sheet and an alpha helix which interact via a zinc molecule.


what is the general structure of a zinc finger domain?

3 zinc finger motifs that each bind to 3 nucleotides.


how is the zinc finger domain transformed into a zinc finger nuclease?

you attach a fok1 which is a restriction endonuclease


what is the best way to improve binding specificity of gene editing tools?

have two different binding sites either side of the target site which then interact to cause cutting


what is the general structure of a zinc finger endonuclease tool?

two zinc finger domains that are attached to a split fok1. each interacting with 9 nucleotides either side of the target site on opposite strands.


what, in theory, is the process by which zinc fingers can be constructed in relation to the base pairs they interact with and why is this not true?

you have different zinc finger motifs for each triplet and you can put three motifs together depending on the three triplets you want it to bind to. This is not the case as each finger can alter the binding specificity of the other . This is because they can reach across and interact with the major and minor grooves to influence binding to the DNA and interactions with each other


how do the three zinc finger motifs interact to complicate binding specificity?

they can reach across and interact with the major and minor grooves to influence binding to the DNA and interactions with each other


Because of the complicated interactions that the zinc fingers have with each other and the complications that this can have on binding specificity, how can you ensure that your zinc finger is right/

you have to undertake a lot of screening and selection. methods include OPEN, CODA and alternate zinc finger selection strategy.


what are the 3 screening strategies for zinc fingers?

OPEN, CODA and 2 other random alternate zinc finger selection strategy.


what is the Oligomerized pool engineering selection mechanism for selecting the right zinc finger domain?

oligomerized pool engineering (open) selection method: this involves having finger pools- contains vectors containing genes for sifferen zinc finger motifs- you can then use primers to amply a random finger from each pool- they have complimentray ends and begininngs so that the fingers can anneal and act as primers for each other. Then you have a random triplet ZFD, then this is cloned into a B2H phagemid and expressed as a fusion protein with Gal11p. This is then put into a B2H selection strain(e.coli). This selection strain contains a ZFN “half-site” positioned upstream of a B2H promoter which drives expresion of selectable markere genes and a hybrid alpha gal4 which acts as an RNA polymerase. To make this strain the target site is cloned into a plasmid and use homologous recomb to clone into the site and mating of the e.coli to pass along the target site. When the phagemid infects the e.coli, the expression of a zinc finger domain that binds to the target site will bring the Gal11p next to the hybrid alpha protein Gal4 and drive the expression of the marker genes- the e.coli colonies expressing the reporters can then be selected and teh zinf fingers extractaed and anylised.


how could you slightly alter the OPEN selection protocol?

you could use the ZFD to drive transcription of a resistance gene instead of a reporter and grow the cells on something that causes cell death (neomycin?)


what is the CODA selection process for zinc finger domains?

This uses known zinc fingers for targeting known triplets. For example, if you have a ZFD which targets successful the first 6 nucleotides that you are interested in and you know another ZFD that successfully targets the last 6 nucleotides of your site of interest, and they both share the second finger, then it is likely that the F1 from your first and the F3 from your second will interact well together with the shared F2 in the middle. You can produce this using Primers and PCR for each finger with overlapping ends which act as primers for each other- construct the three finger domain and it is done


what are the draw backs of the CODA selection process?

- Only works for about 2.55% of all possible 9-pair sequences (only this amount of genes have known common F2s that will ensure complimentary ZFD)- Mostly works with triplets that have the code 5’-GNNGNNGNN-3’. Thus, can target only about 81% of genes (not sequences) in fish 63% of all genes in Arabidopsis


what is the success rate of using CODA to target zebrafish genes?

there is a 50% success rate


describe the 2 other random inc finger selection strategies

1. have a zinc finger library and randomly sticking zinc fingers together and then you have a target site that you want to target and you have an out of frame GFP on the right, a stop codon at the end of target site and an GFP on the left. If the zinc finger binds and allows for cutting- you will break this stop and it will cause the cell to express GFP. This construct would be injected into cells previously. 2. take a library of zinc fingers and randomly put triplets together to create zinc finger domains and put each triplet into a construct with a Fok1+ and then another with a Fok1- (so the two halves of the endonuclease). These two constructs also both express a specific marker gene. Then you construct a plasmid which expresses two marker genes: one is between the two target site of interest and one is not. You then infect a yeast with these three plasmids (that all have promoters upstream of the ZFD and upstream of trapped reporter. If the zinc fingers both target the trapped gene then the trapped reporter will be cut. You then select for yeast colonies that express the three reporters but not the trapped one. You put combinations different plates and then keep track of which combinations in which plate so you can refernce which ones are successful.


what are the drawbacks of zinc fingers?

want to be able to target any sequence but you have to screen for the correct sequences etc and certain zinc fingers can hinder each other etc so have to check all this before and might not work for the sequence that you want cut rate is fairly low


what needs to be considered when testing zinc finger domains in vitro?

this may be no representation of how they may work in vivo- how sensitive they will be heterochromatin


what were TALE proteins naturaly used for?

bacteria secrete these into plants (repeating structures that bind the DNA- and activate transcription of genes- nutrient genes that make it easier form the bacteria to get in and infect it)


what is the general structure of TALENS?

They are repeating units, each modular unit is identical bar a diamino acid structure which binds specifically to a nucleotide. They have increased specificity due to their ability to bind to around 14 base pairs either side of the target site.


what needs to be considered and are potential down sides to using TALENS?

- they dont bind as tightly as ZFN- they are very laborious to make: the repeating structures mean that they can recombine within bacteria when they are being cloned. so you have to use bacteria that are recombination deficient but these have their problems too.- They are generally very tedious to put together


what are the three method of TALEN construction?

FLASH assembly, golden gate cloning or standard restriction digest.


what are the areas of the TALEN modules which are repeated, called?

repeat variable diresidues.


what is the flash assembly of TALENs?

- a dna fragment encoding a single TALE repeat is labelled on its 5’ end with biotin and is initially ligated to a second DNA fragment encoding 4 specific TALE repeats and then attached to a streptavidin-coated magnetic bead. Additional DNA fragments encoding pre-assembled ATLE repeates are ligated until an array of the desired length is assmbled. The DNA fragment is then removed from the bead by restriction digestion which removes the biotin bead.


what is the golden gate cloning process of TALEN construction?

You use PCR to amply each unit, you may have for example 6 in each pool. You then add appropriate ligation adaptors. so that each of the units will ligate to the next one along although the 1st and the 6th will not ligate together instead they will have the same restriction site, but there will be a spacer complimentary site annealing site. Then, using the intermeidate space between the 1st and 9th, the hexamers are amplified and purified. The next step involves cutting the hidden sites at after the 1st and 6th and then 6th restriction site is then revealed which will then attach to the 7th start. and the same for the 12 to the 13th. Then they are put into a cloning backbone which may be a viral vector which then fuses it to a functional domain. this is done by ligating the backbone with the same restriciton enzyme that is at either end of the TALEN units.


what is the standard cloning based method?

making two modules at a time and fusing them using standard restriction enzyme digestions and ligations. Then these two modules are fused to each to form 4 modules. then the 4 modules are fused together etc etc


are zinc fingers good with methylation?