deck_4988184 Flashcards
(110 cards)
1
Q
what are the general aims of someone wanting to edit a genome?
A
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
2
Q
what are the two response to double strand breaks?
A
non-homologue end going or homologous recombination
3
Q
what normally happens when double strand breaks are repaired by no homologues end joining?
A
mistakes are made by the repair machinery and point mutations, deletions, insertions which causee frameshift and maybe truncated protein (KO)
4
Q
what are the three main genome editing tools used?
A
zinc finger nucleases, CRISPR/Cas9, TALENS
5
Q
what is the general structure of a zinc finger motif?
A
a 2 strand beta sheet and an alpha helix which interact via a zinc molecule.
6
Q
what is the general structure of a zinc finger domain?
A
3 zinc finger motifs that each bind to 3 nucleotides.
7
Q
how is the zinc finger domain transformed into a zinc finger nuclease?
A
you attach a fok1 which is a restriction endonuclease
8
Q
what is the best way to improve binding specificity of gene editing tools?
A
have two different binding sites either side of the target site which then interact to cause cutting
9
Q
what is the general structure of a zinc finger endonuclease tool?
A
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.
10
Q
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?
A
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
11
Q
how do the three zinc finger motifs interact to complicate binding specificity?
A
they can reach across and interact with the major and minor grooves to influence binding to the DNA and interactions with each other
12
Q
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/
A
you have to undertake a lot of screening and selection. methods include OPEN, CODA and alternate zinc finger selection strategy.
13
Q
what are the 3 screening strategies for zinc fingers?
A
OPEN, CODA and 2 other random alternate zinc finger selection strategy.
14
Q
what is the Oligomerized pool engineering selection mechanism for selecting the right zinc finger domain?
A
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.
15
Q
how could you slightly alter the OPEN selection protocol?
A
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?)
16
Q
what is the CODA selection process for zinc finger domains?
A
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
17
Q
what are the draw backs of the CODA selection process?
A
- 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
18
Q
what is the success rate of using CODA to target zebrafish genes?
A
there is a 50% success rate
19
Q
describe the 2 other random inc finger selection strategies
A
- 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.
20
Q
what are the drawbacks of zinc fingers?
A
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
21
Q
what needs to be considered when testing zinc finger domains in vitro?
A
this may be no representation of how they may work in vivo- how sensitive they will be heterochromatin
22
Q
what were TALE proteins naturaly used for?
A
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)
23
Q
what is the general structure of TALENS?
A
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.
24
Q
what needs to be considered and are potential down sides to using TALENS?
A
- 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
25
what are the three method of TALEN construction?
FLASH assembly, golden gate cloning or standard restriction digest.
26
what are the areas of the TALEN modules which are repeated, called?
repeat variable diresidues.
27
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.
28
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.
29
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
30
are zinc fingers good with methylation?
no
31
does the efficiency of the TALEN cutting decrease when there are more methylation sites?
yes
32
when there are around 4 methylation sites, what is the percentage cutting of the TALEN?
40-50%
33
how can you use TALE to modulate the methylation of a DNA site?
you can construct the same DNA targeting mechanism but then instead of attaching a for 1 you can instead attach a DNA methyltransferases (DNMT). This then methylates the DNA and turns it off. This would mean that you would follow the same procedure to produce the TALE part but when you ligate it into the cloning backbone you would fuse it to a DMT rather than a Fok.
34
how can you use TALE to modulate the acetylation of a histone at a DNA site?
You you can construct the same DNA targeting mechanism but then instead of attaching a for 1 you can instead attach "histone acetyltransferase" (HAT) or "histone deacetylase" (HDAC). This will either unwind the DNA or cause it wind by adding an acetyl group to the histones associated to the DNA
35
what are the pitfalls of TALENS?
modular build is very tedious and long winded, they are very big constructs, not all sites are equally well targeted.
36
what was the CRISPR/Cas9 originally used for?
bacteria used it as a mechanism to capture viral DNA and store it as a memory so that they could then chop up the DNA if it entered again.
37
what is the structure of the DNA target sequence of the guide strand within the Cas9?
The content of the guiding sequence should have a GC content of 40%-80%, the length and should be 17-24 (smaller = less of target effects)
38
how does the CRISPR/Cas9 sequence work within bacteria?
The area of the genome encoding the CRISPR looks as follows: - the tracrRNA followed by a CAS operon which encodes different CAS enzymes, followed by a cRISP repeat spacer array which contains genes encoding the viral DNA with spacers in between. When this gets transcribed, the tracrRNA binds with the Cas9 and allows the CAs9 in conjunction with RNAaseIII cleve at each spacer and produce mature crRNA. This results in an RNA duplex consisting of the tracrRNA and the mature guide RNA which is then used by Cas9 to cleave the target DNA in two places on each strand using two domains of enzymatic activity
39
how did people take the matures structure of the guide tracrRNA and crRNA duplex and use it to create a more efficient guide?
They looked at the tracrRNA crRNA duplex and realised that if you add a linker between the two then you can create a single guide strand that can be provided to the Cas9 enzyme. this is called a single guide RNA The tracrRNA has different hairpin loops
40
what is the structure of the tracrRNA?
has hair pin loops
41
what is the difference between the specificity of the Cas9 system compared to the ZFN and TALENS? what does mean for the application?
there is only one guide and so you lose the specificity with Cas9
42
describe the experiment which looked at the specificity of the CAs9 system
create cell that that expressed short lived GFP gene which, when broken by a double stranded break, will result in the cell losing its GFp expressiosn They designed missmatches in the site and guide and asked how efficient was the Cas9 and guide to cut at the site of the GFP. They showed there could be up to 2-3 mismatches in the bases between the target and the guide and you still get efficient cutting.
43
why does the Cas9 system naturally not want to be too specific?
because bacteria want to be able to recognised evolved viral DNA
44
what is a Cas9 nickase? how is it formed?
it only cleaves one strand of the DNA. It is formed by mutating the Cas9 protein
45
how can Cas9 nickases be used to produce increase Cas9 specificity?
you can use two nickases which two guides that target either end of the target site, one cleaving one strand and one the other.
46
how is the spacing important with nickases?
if you have the two pairs too far apart then it doesn't work
47
what is the guide called for Cas9?
sgRNA (single guide RNA)
48
what is the PAM site?
it is a specific sequence that has to be targeted at the end of the guide (3' end of the guide but on opposite target strand) and for the classical cas9 this is NGG. this allows for the proper alignment between the guide and the double strand and the loop structure. You need a 5' to 3' sequence that ends in NGG
49
how are people making increased specificity Cas9?
by mutating the Cas9 protein
50
because Cas9 guides can have off target effects, what is an experiment that you can do to ensure that the mutation that is produced following a Cas9 approach is targeting your desired sequence?
you use one guide that has known off-target sites A,B or C. Then you have another guide which has the off-target sites D,E,F. you use both on different organisms then you get a phenotype. Then you can cross these and see if you still get a phenotype (compliment testing ). if the phenotype is being cause by cuts at two different sites then a het will be formed when crossed and you will lose the phenotype- but if you dont lose phenotype then it is at the same site (fail to complement) 2. do the two guides give the same phenotype- if they do then likely they are not targeting the same gene 3. you can outcross you animals to clean them up
51
what is the PAM site for classical Cas9s?
5'--> 3' NGG
52
how much of the human reference genome contains GG? what does this means about how many target sites there are per how many bases?
5.21%, 1 target site every 42 bases
53
describe a selection experiment that can be used to produce mutant Cas9 proteins that require a different PAM sequence.
mutate Cas9, put into selection screen by using a plasmic containing Cas9 under the control of a T7 promoter then a sgRNA under teh control of a T7 promoter followed by a resistance gene for selection. Then you also have a plasmid which has your target site that doesn’t have a GG downstream of a toxic gene target site that when cleaved will allow growth. An example of a toxic gene would be arabinose-inducible toxic gene. Then the bacteria can be plated in the arabinose and only those bacteria colonies containing Cas9 mutants that can cleave the mutant PAM will allow the colony to grow. Then you can make sure that the Cas9 is specific to that Pam sequence and not others but selecting those mutants which will cut a certain PAM sequence but then use the positive selection screen to test whether, when you mutate the PAM back to NGG, does it still work. If not then that is good! The positive selection screen is done by using target site containing a resistance gene is upstream of a NGG. If the PAM is refractory to the Cas9 then the colony will be allowed to grow when grown of a medium with the compound requiring resistance against. As long as you keep track of which Cas9 mutants you are using you have work out which work with specific pam sites and not with others.
54
describe an experiment which will allow you determine what makes a good gRNA for CRISPR
CRISPR scan- they have taken different gRNAs for different sites within the same gene and asked what was the cut rate was for each gRNA target. and then they looked for rules within the guide which were determining what makes a good guide.
55
how can you use the Cas9 system to silence transcription?
once you can target a certain area you can add enzymes which activate transcription. You can mutate the nickase sites of the Cas9 so that it isnt actually cutting, instead it will just bring a fused protein. You can use this to silence ranscription by using a enzyamtically dead Cas9 that will bind to a DNA region and will interact with the PAM sequence so that ranscription is haulted when ti reaches this point.
56
how could you produce a light sensitive cas9 and why would you do this?
You can put a light sensing domain on the Cas9 which will allow it to be taken into the nucleus and then out again. This could be used to spatially or temporally control transcription. you can do this by using a light sensitive nucleus localising signal which is a protein that could be fused to a protein and is based on the LOV protein domain- this could be used to activate or suppress transcription when you wanted.
57
how could you use crispr cas9 to report the transcription of a mutant gene?
inject into the early embryo, a cas9 which a guide RNA for an area downstream of a enhancer r promoter. Then also inject a plasmid which contains homologous arms and a reporter gene with a stop codon. When the cas9 cuts, this will be inserted and stop transcription of the protein but will express the reporter. this can be used in a gene trap way
58
are crisprs efficient ?
yes
59
how can you perform a Cas9 genome wide-esque screen.
inject a pool of guides targeting different genes into the embryo with the cas9 or into an embryo which already expressed the cas9. And then look for a certain phenotype. Then you can identify which guides were injected into this pool then do individual screen to find which gene was being targeted
60
in what organism is a cas9 screen should e carried out in?
zebrafish because can be injected early into the embryo and will start cutting early
61
describe how the gene drive works.
you target a gene with a construct that encodes both the cas9 and the guide RNA, for itself this is inserted into the gene on one of the chromosomes this is then transcribed and the crispr cas9 forms a KO in the other chromosome because of the homology on either side of the CAs9 on te other chomosome, homolgous recombination results in the cas9 construct recombining into the other chromosomethis means that any cell that expresses this construct will be homozygous this means that when you cross a male whci expresses this and you get a “het” offspring, all cells will then become homo because the cas 9 construct will attack the other in most casesthis means that instead of getting a mednialin ratio you can get all offspring eventually expressing as there is an over 50% chance of inheriitng it. if you target a gene involves in sterility you can knock out an entire species.
62
how can you detect mutant events in zebrafish?
you inject crispr/cas9 stuff into the embryos. You can't screen embryos and then put them back. SO you screen half of the clutch you injected and then use DNA melt of Primer PCR to identify a mutant event. You then infer that the other 50% has been equally successful.
63
what is the high resolution melt curve and what is it used for?
it is an efficient way to ID mutants, you melt the DNA and differentt DNA sequences melt at different times do you can identify changes within a certain region of DNA by comparing to wild type
64
what are the problems with using th melt curve in zebrafish?
you dont have homogenous lines in zebrafish so need to watch out for SNPs.
65
how can you sequence mutants and ID the type of mutation is a way that is better than melting? why is this better?
You can PCR region of interest and then deep sequence many different fish. This will allow you identify different mutant loci and identify which one you want. then you keep the fish that contains these mutations and breed this one as a strain
66
what type of errors can non homologous end joining result in?
insertions, deletions, substitutions at the break site
67
when can non homologous end going mediate translocation?
when there are two double stranded breaks then the ends can be swapped when repaire .
68
what is the process following breakage of DNA that stimulates DNA repair?
Following the detection of a DSB, the histone variant H2AX is targeted for phosphorylation in chromatin close to the break site. The phosphorylated form of H2AX, known as gH2AX, serves as a molecular beacon, signaling the presence of damage by marking nucleosomes in one or more megabases of DNA surrounding the DSB (Rogakou et al., 1998). gH2AX is central in linking damaged chromatin to the DNA repair machinery, directing the recruitment of multiple DNA repair and signaling proteins into repair centers, microscopically visible nuclear aggregates known as ‘‘foci.’’
69
do the last two lectures!!
...
70
what are the enzymes that clear DNA?
nucleases or endonucleases
71
in what organisms have TALENs been used?
flies, yeast, zebrafish, rat, tips cells and human somatic cells.
72
why are TALENS good to use in relation of the construction?
they rely on a repeated domains that only change for the two amino acids that give them their base pairs specificity- these are two residues at position 12 and 13
73
has the Binding efficient of ZFN and TALENS been directly compared?
no but studies have shown that they can cleavage specific genes with similar efficiency.
74
where are the 2 varying residues in the TALEN modules?
usually position 12 and 13
75
what do the the two hyper variable resides of the TALEN modules interact with upon DNA binding?
positioned in the DNA major groove
76
what are the names of the modules of the TALENs and what is each module's base pair?
NN= guanine NI= adenine HD= cytosine NG= thymine
77
once you have made a double strand break, what can you do?
mediate homologous end joining or allow the DNA repair machinery to make mistakes and introduce mutations.
78
Do TALENS have a high success rate?
one study said that they did have a high success rate in any DNA sequence of interest that was performed in human cells.
79
how can you check if you mutation has been successful?
All of the mentioned techniques require that you amplify up using PCR your target region, you can do this because you know the site around the mutation and you can use primers. 1. you can insert your PCR product into a plasmid vector that contains a reporter gene. impairement go recovery of the reading frame of the acZ gene occurs via insertions or deletions- you can then see how many colonies exrpress the reporter from each PCR product 2. you can use Sanger sequencing your PCR product 3.use heteroduplex formation and then electrophoresisform heteroduplexes then use high resolution melt curves
80
what is the heteroduplex process of analysis if a mutation has been induced?
because you obviously know the surrounding region of your gene due to targetting it, you can make primer amplifying the target locus once you have extracted the DNA and used restriction enzymes. Once you have the PCR product you can mix this mutant DNA with wild type DNA and then denature and induce hybridisation- this will form a heterodulpex which is retarded in electrophoresis- can determine the presence of mutations
81
what is sanger sequencing?
During Sanger sequencing, DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain (extension product). Chain elongation occurs at the 3' end of a primer, an oligonucleotide that anneals to the template. The deoxynucleotides added to the extension product are selected by base-pair matching to the template.The extension product grows by the formation of a phosphodiester bridge between the 3'-hydroxyl group on the primer and the 5'-phosphate group of the incoming deoxynucleotide (Watson et al. 1987). Growth occurs in the 5' -> 3' direction (Figure 1).DNA polymerases can also incorporate analogues of nucleotide bases. The dideoxy method of DNA sequencing developed by Sanger et al. 1977 takes advantage of this characteristic by using 2',3'-dideoxynucleotides as substrates. When dideoxynucleotides are incorporated at the 3' end of the growing chain, chain elongation is terminated selectively at A, C, G, or T. This is because once the dideoxynucleotide is incorporated, the chain lacks a 3'-hydroxyl group so further elongation of the chain is prevented. you then do electrophoreses and the labelled terminating bases will read out downwards giving the sequence
82
how can you induce large deletions using gene editing tools?
target two sites surrounding a region
83
how can genome editing be used in disease study?
you can use these techqniues to target genes involved in diseases- such as dystrophin and then create a disease model that you can work with
84
which of the TALEN making mechanisms has the highest throughput?
solid phase
85
give some precise data demonstrating the superiority of TALEN cutting to ZFD. what organism?
85% of the 32 TALEN pairs used induced somatic indeed stations but only 25% of the 84 ZFD used induced indeed mutations (ZFD were made from the CODA technique) in zebrafish
86
which gene editing tool has been shown to be sensitive to CpG sites?
TALENs - more than 2 or site sites decrease mutagenicity
87
how can the sensitivity of talens to CPG sites be circumvented?
can use a N* module rather HD module
88
when can TALEN mutations be inherited?
if injected into the embryo early then they can enter the germ line
89
how can TALENs, CRISPR and ZFD be induced into the entire embryo and what embryos can this method be used in?
If you inject the mRNA of the TALEN, ZFD, or CAS9 sgRNA into a single cell embryo. You can also inject lentiviruses expressing the construct. You can inject the TALEN mRNA into the mouse male pronucleus.
90
what is the problem with the fok1 dimerisation theory? and how has it been circumvented?
you can separate the uncle domain must homodimerise to function. sometimes the foc1 domain can homodimerise when only one monomer binds to DNA and can produce off target effects. Instead you can create a fok1 that must heterodimerise- this reduced off target effects
91
what is a big downside of ZFN and an issue that comes with this?
The binding of the 3 motifs are not always complimenty to each other so it requires a lot of screening to check the zinc ginger will work. it is hard to produce high activity zinc finger domains that are not cytotoxic
92
what is the problem with he resources available for ZFN?
there isn't a library containing 64 inc fingers all combinations of the triplet sites
93
what are the criteria for a good zinc finger target site? compared to talens?
guanine rich and have the sequence GNN, talent can target any site
94
what is the only apparent restriction of TALEN target site?
a thymine at a the 5' end of the target sequence
95
how can TALENS be modified so that they can recognise methylated DNA?
the requirement for TALENS is to have a thymine at the 5' end of the target sequence but a However, a methylated cytosine is indistinguishable from a thymine in the major groove; hence, the His-Asp RVD repeat (which recognizes cytosines) can be replaced with an Asn-Gly RVD repeat (which recognizes thymines) to generate TALENs that can cleave methylated DNA
96
what does pam stand for?
photospace adjacent motif
97
how can you find cas9 proteins that recognise different pam sequences? (2)
1. by carrying out mutant cas9 screens 2. using cases from different species
98
what is a major advantage go crispr over talens and zinc fingers?
they are extremely easy to prepare- just need to make the sgRNa
99
what are the requirements for the cas9 guide?
17-23 bp long, 5-NGG-3 pam sequence after it. 5' end start has a guanine
100
why is a guanine needed at the 5' end of the cas9 guide? how can be circumvented?
because guide RNAs are transcribed by RNA polymerase III under the control of the U6 pro- moter in cells; therefore, RNA transcripts with bases other than guanine at the 5ʹ end are poorly transcribed. circumvented by making guide RNAs with one or two additional guanine bases at their 5ʹ ends
101
describe te cleavage efficiency of the Cas9?
Unlike ZFNs and TALENs, RGENs can cleave methylated DNA, but not all sequences that contain the PAM sequence are cleaved efficiently by RGENs in cells. Understanding the molecular basis for this occasional failure is currently an active area of research.
102
compare and contrast the success rate of talen and crispr and ZFN
Not all newly synthesized nucleases are functional and equally efficient. Many ZFNs, especially those created by modular assembly, fail to cleave chromosomal DNA in cultured cells or in whole organisms. Despite continuous improvements in ZFN technology by academic researchers, com- mercially available ZFNs generally work better than ZFNs created in the laboratory using do-it-yourself kits. By contrast, TALENs have a nearly 100% sucess rate in mammalian cells if one avoids heavily meth- ylated target sites15. Nonetheless, mutation frequencies obtained with functional TALENs range from 1% to ~60% in mammalian cells. Similarly, RGENs have a broad range of genome editing activities (2.3–79%) in cultured mammalian cells. The success rate and activity of the three classes of nucleases depend heav- ily on the cell type and delivery method. So far, there are no reliable rules to predict nuclease activity before experimental validation.
103
what are the pros and cons of making a long TALEN target site?
- a long time to construct - more specific - can cause off target effects due to partial binding
104
how long ideally would a talen be to cause no off target effects?
16 bps
105
which gene editing techqniue is known for its cytotoxicity due to off target effects?
ZFN
106
what is the danger of off target effects?
transclocations
107
how do the three gene editing tools vary in their mutational signature?
Although ZFNs and TALENs share the same FokI nuclease domain, their mutation patterns are surprisingly different. A comprehensive survey of indels induced through error-prone NHEJ in cells and whole organisms showed that, unlike TALENs that pro- duce deletions much more frequently than insertions (89% versus 1.6% in mammalian cells), ZFNs induce insertions and deletions at comparable frequencies124. ZFNs produce defined four- or five-nucleotide 5ʹ over- hangs that are filled in before NHEJ, which frequently yields small insertions. Interestingly, one- or two- nucleotide insertions are often obtained at RGEN target sites5. In line with this observation, RGENs occasionally produce one- or two-nucleotide overhangs when they cleave DNA in vitro, in addition to the more prevalent blunt ends115,125 (FIG. 4c). Defined overhangs are often use- ful for targeted insertions of plasmid DNA at specific sites through NHEJ22.
108
discuss the methods tht can be used for the introduction of the gene editing machinery into animals.
- plasmid DNA injection - in vitro transcribed mRNA- injected into the embryo at the first cell stage. can lead to faster expression com[ared to DNA plasmid and unwanted integration of the dna in the genome - viral vectors : non integrating such as AAVs and integrase-deficient lentivirus vectors. ZFDs are best for this due to short length. TALENs not good if IDLV because of their repeated structures that can induce recombination. adenoviruses good because large cargo size - integrating viruses such as lentivirus for crispr/cas9. - purified proteins- inject proteins int cells that are difficult to transfect - electroporation of DNA- used in cell lines
109
what is the biotin labelled DNA fragment attached to during the FLASH TALEN assembly?
stretavidin-coated magentic bead.
110
how could you temporally repress or activate transcription using the gene editing techniques?
by using a light sensitive nucleus localising signal which is a protein that could be fused to a protein and is based on the LOV protein domain- this could be used to activate or suppress transcription when you wanted. You can fuse this to a TALE that has had its endonuclease domain removed and fused with another protein
