Cpf1-based genome editing using ribonucleoprotein complexes

and welcome to this integrated DNA technologies webinar on genome editing with the alter CRISPR CPF one system my name is dr. Han speaker and I'll be serving as the moderator for today's presentation the presentation today will be given by dr. Ralph Turk Ralph is a staff scientist here in the melodic molecular genetics Applied Research Group at IDT where he has been working on a variety of projects including optimization of delivery protocols for oligonucleotide protein and the altered CRISPR systems dr. Turk received his PhD in molecular genetics from the University of Leiden in the Netherlands where he studied disease mechanisms underlying muscular dystrophy using high-throughput methodologies Rallis presentation today should last about 30 minutes and following that presentation Ralph I answer your questions as attendees you've been muted but we encourage you to ask your questions at any time during the presentation and you can do those by typing them into the questions box located in the GoToWebinar control panel and you can actually so there's a little down arrow you can make it bigger there in the panel or you can pop it out using a little plus sign or up arrow that's on the right-hand side of that bar and your interface and make that even larger to take this question into it make it nice and easy for you also in case you need to leave early today we are recording the presentation and we will make the recording available on our Vimeo channel and our YouTube channels searchable on the screen you don't have to remember this we'll also send you links to this after after the webinar in the next couple of days with links to the to the videos and the slides the slides we posted during the presentation today probably and those will be on SlideShare net /id t DNA and those would be available pretty soon all right and I guess with that we can get started with the actual presentation so indeed like I was saying and thank you for the introduction and I'm going to talk about CP of one base genome editing using the Eldar CRISPR CP of one system so this is a quick outline of what I'm going to talk about today first I'm going to give you a background then I will move on to the optimization of the CP of 1c RNA or crispr RNA we or have optimized both the length and the chemical modifications for this crispr RNA to make it more stable and more effective leading to higher editing efficiencies then I will move on in the delivery of the CPF one as a ribonucleoprotein complex so that is where the CP of one protein is complex together with the crispr RNA and delivered directly into cells as a rfp complex I will talk about the effect of the RCP upon electroporation enhancer which is a single-stranded oligonucleotide that will or that does increase the editing efficiencies I will talk a little bit about the RMP concentration optimization so you can kind of dial down maybe the amount of RMP that you have to use in your genome editing experiments at the end I will show a couple of slides about homology directed repair using CPF 1 and positive controls that we have available for this system so genome editing basically revolves around the generation of double-stranded breaks in genomic DNA and this is done by specific enzymes most famously Kaz 9 and now what I'm going to talk about CPF 1 and these proteins are what are called RNA guided endonucleases and they cut genomic DNA at very specific locations and that specificity is generated by the crispr RNA that is combined with these with these proteins so upon cleavage of the genomic DNA a couple of different pathways can be activated in the in the cell the most prevalent one is the non-homologous end joining pathway mhm aj as you can see over here and what that will lead to often are mutations and deletions or mutations in the form of insertions or deletions and if you're for instance targeting an exon this can lead to a out of frame transcript and thereby render the protein that you're targeting not not functional the other thing and the other pathway that can be activated is what is called the homology directed repair and what you need for that is the presence of a template strand that has homology arms to the cut site and that is what is represented over here in light blue that is the those are the homology arms you can introduce your template either as a double strand template or a single strand template and we like you to use the single strand template because the chances of blunt ended or insertion of your template is minimized but we will have other webinars talking about that in the future so let's start off with an overview quickly of the cast nine system so cast nine is a RNA guided and the nuclease like I said before it relies on binding to a guide RNA which consists of a tracer are in a complex that binds directly to cast nine and then a crispr RNA molecule that binds to tracer RNA and the CRISPR molecule contains a what is called a proto spacer sequence and that is what generates the sequence specificity where the cut is being generated in your genomic DNA and that is about 20 nucleotides in length the binding of this RMP relies on the presence of a pam site or a proto spacer adjacent motif in the case of cast 9 it is mgg so in order for the double-stranded break which cast 9 makes as a blunt and at site you need to have the presence of the ngg and then your sequence with specificity of your produce pacer so CP f1 is a different RNA guided and in the case and differs a little bit from cache line it stands for crisper from Prevotella and Francis lo1 but these are two bacterial strains where the dispersing was found initially it's a class 2 type 5 RNA guided and anaphase and upon testing the the CPF protein derived from either Prophet Allah or Francis Allah it was found that the editing that could be achieved in mammalian cells was rather low so the group of van tested a bunch of different CPF proteins from different strains and found that especially the CPF one protein from a seed amino caucus elect mysteriously was active in mammalian cells and that is why we chose this a s or a state amino caucus version of CPF 1cp Vaughn also differs from Kaz 9 in that it uses only a single guide RNA so there's no tracer RNA the CR RNA binds directly to the protein and is usually between 41 and forty four nucleotides in length also different from Kaz 9 it generates a double-stranded break with staggered and so there there are five prime overhangs and the other way that CP of one differs is that it uses a thymine rich pam side and it preferentially uses TTT V so these are the pam sides that are recognized titi ta-a C and G and we have some data later on in the press in the presentation where we show that specificity as as well so the group of osama Nawrocki published the crystal structure of CP of one where it is bound to genomic DNA as well as its crispr RNA and you can see the crystal structure depicted Oh here there are a couple of domains across this protein that lead to the actual cleavage so over here they made a really nice schematic representation of the interaction of CP of one CR RNA and double-stranded DNA template to to be cut and what you can see over here is that on one lobe a cut is generated by the rough sea domain whereas another cut is created by the nook domain now what is interesting too about CP of one is that this cut on the non target strand needs to occur before the second cut can be generated and that is where it differs to so again just to wrap up this this background a little bit casts nine the most famous one so far uses two part guide RNA the tracer RNA and a CRNA they can be combined by a linger to create a single guide RNA and this is something that I talked about in in one of my previous webinars which by the way you can find at our IDT website so the other thing is that cache cuts bluntly and compared to CP of one where there are five prime staggered ends again there's a single guide RNA present over here so the reason why we have CP of one as a product is that it does differ a little bit from Kaz nine if you for instance have a genomic region that you want to target that is very eighty rich you might have more success in finding a proper target sequence using CP f1 and versus caste because nine also there are parentheses a couple of different organisms such as a Plasmodium falciparum that is causing malaria that is that has very ap rich genomes there's a couple of other benefits to the CP of once that will go over in the next couple of slides so the group of Ginsu kim published a great paper in Nature Biotechnology talking about the specificities of CP f1 and the new phases and one of the things that they did was comparing the on target and off target efficiency of CP upon compared to cask as night so they in this case looked at a couple of different targets they have a figure in the paper which is shown over here where they look at the on target effect of this specific target site and that is what you can see here and the and the length of this bar represent the editing efficiency and what you can see that overall the two strains so that's a see them in a caucus CP of one and like no Spira see CP of one also generate the same amount of editing efficiency so the on target editing efficiency can be the same with CP of one compared to the past nine what are a diverse though is that the amount of off target effects which are represented right over here all across the genome is far less using the CP of one proteins and that is definitely easier to do to see when you take another target site which is dnmt1 - for where the amount of off targets using a s or lb CP of one is very low compared to Cassadine one of the earlier webinars that it gave discuss the use of our in piece of ribonucleoprotein complexes and how it's beneficial when you want to do genome editing because it's quick it's easy and one of the big things is that the amount of off target effects is significantly lower and this is because the turnover of the RMP is is is much high compared to expression say through a plasmid where the time that CP f1 or the TP one are in P lingers around is a lot longer and thereby increases the chances for generating off target effects the same data was shown in that same paper from Jin Zhu Kim's group where they looked at the editing efficiencies both on target of a particular site and compared the RMP genome editing efficiency versus genome editing efficiency when CB of one is encoded through a plasmid and found that they are fairly much the same note that this is a logarithmic scale now when they looked at different off target sites they showed that the amount of editing on these off target site is significantly higher when CPM one is expressed from a plasmid again indicating that the use of an RMP is beneficial over the use of plasmid now one of the things that we looked at here at IDT is how efficient the CP of one new places are so in this case we did what we refer to as gene locks where we take H and Omega region in this case the step 3 gene where we looked at exons 5 and 6 and look at genome editing at every possible target site within that region and this is what you can see over here where we have the chromosome location shown in orange we have all the target sites for cast 9 in blue we have all the target sites for CP f1 and this gives you an idea of how well it is dispersed throughout this this region one thing that you have to note is that the amount of target sites different between caz 9 and C P of 1 and that is because the chances of finding a PEM site of three bases in the case of gas 9 and GG is far more frequent than finding a PEM site made out of four bases which is TD T V or TTP and for CP f 1 so the amount of target sites can definitely differ between the two different systems and is less for CP of one compared to Kaz 9 now if we take these results over here and just plot them ranked by editing efficiency you can see a couple of things for the COS 9 protein which is kind of the standard we can see that the vast majority of the crisper RNAs generate editing results of more than fifteen percent so ninety-three percent of the CR RNAs that we tested generate good editing efficiency now for CP f1 when we looked at every potential target site we found that the amount of CRNAs that are functional is far less so in this case at thirty five percent excuse me now one of the things that I talked about during the background is that the sequence specificity when it comes to the PEM site is pretty particular when it comes to CP of one so I kind of plotted the results of 232 CR RNAs that we checked across six six genes and this is the editing efficiency and I separated them based on their Pam site and what you can see if you have a PEM site that consists of a tttt the editing efficiency is generally very low for this website if you have a PEM site that consists of an ASE or a G the editing efficiencies are a lot higher so for instance over here I plotted the same figure as I shown two slides go where you have the cash nine results and here you have the TP TN from CPM one if you just look at the target sites that have either an an ASC or G and that is referred to as a V here so the TTT V Pam asides you can definitely increase the ratio of successful CR RNA so in this case we find that one and two T RNAs are functional now these are by the way introduced into the heck 293 cell lines as an as an as an RMP using the Maxima the effector system and I will go a little bit more into that in the further down in the talk and and and how we optimize that but overall we at this point recommend to if you design your target sites only to look at the TD T V Prem site now over here is a schematic again of CP of one protein combined with the CR RNA and how its interacting with the double-stranded DNA and where the cuts occur one of the things that we did was try to optimize the CR RNA to ensure that the editing efficiency was as high as as possible so one of the things that we did was we did a length optimisation of either the constant region which is over here and this is the region that binds to CP of one protein and we did an optimization of the proto spacer region and that is the region that generates the specificity now when we truncated the constant region even with one base we saw a huge drop in editing efficiency indicating that this constant region cannot be truncated whereas with the proto spacer region when we started truncating it from the 3 prime side side onwards found that looking at a couple of different target sites within the HBO T gene we looked at different lengths of this produce spacer and one of the things that we found that if the produce spacer length is truncated up to 21 it's the editing efficiency is generally highest across all these sites and we tested a lot of other sites too but this is a kind of representative data for that now one thing that we didn't show over here but what we studied as well is that if we even decrease the length of the proto space in more to 19 18 and 17 we saw a huge drop in editing efficiency as well indicating that a certain length of the proto spacer is necessary for its first function and we recommend to have a 21 nucleotide long per the spacer the constant region is 20 nucleotides long so the total CR RNA that we can generate and make is 41 nucleotide long the next step that we did when it came to optimization of the CR RNA sequence is to look whether we could stabilize the CR RNA by introduction of two primal methyl groups and this is beneficial in in in two ways because the two primal methyl modifications lead to a it it makes this year RNA less recognizable for endonucleases second of all it does it isn't as easy li recognized by the toll-like receptors so upon introduction into this cell you do not generate a strong innate immune response so what we did we did a couple of walks throughout either the constant region over here or these two primal methyl walks throughout the proto spacer sequence and we found that introduction of these two primal meth site at specific locations for instance over here led to a drop in total editing efficiency indicating that these positions could not be modified and that was the same in the proto spacer sequence and that data is kind of summarized over over here where the length of the bar represents how strongly these particular locations are affected or negatively affected by the two primal methyl modification the other thing that we did was we looked at different other types of chemical modification of the C RNA but because of the proprietary nature of it I cannot show the data but suffice it to say I we we now sell a fully optimized CR RNA that is is that that generates the best editing efficiencies for CP f1 another thing that I want to point out is that if you go to our website we have written a user guide for the Eldar CRISPR CP f1 system where we go into detail how to deliver these RMPs into heck 293 cells using the Emacs and the infection system I would invite you to have a look at that unfortunately due to time I don't cannot go into all the details that that are in there but the information is definitely available so this is a really schematic overview of how easy it is to do the genome editing using the author CRISPR CP of one system and basically what you do is you take your CP of one protein you combine it with your CR RNA for 10 to 20 minutes and that forms the RMP or the interaction of the guide RNA together with the protein to ribonucleoprotein complex so once your complexation is done your this is ready to be introduced into cells using either the perfection or I mean electroporation in general like affection and I'll show some data data later on and or a micro injection of it one of the things that we found was that initially when we tested CPF on protein we found that editing efficiency were generally on the low side compared to caste nine one of the things that I that I worked on was trying to increase that editing efficiency or caste 9 and one of the things that that we now have as a product is called an electroporation enhancer which is a single-stranded oligonucleotide which does not have any homology to either the mouse human or rat genome so you don't have to worry about any micro homology that could generate any nonspecific insertion of the sequence into your cut so sorry into your cut site one second well the same thing we have done with CPF 1 we put in a good amount of effort to find an optimized electroporation enhancer for this RNA guided endo endonuclease so we tested a bunch of different sequences found a particular one that works very very well and the results you can see over over here and this is just example of what it does so we tested the editing efficiency of the CP of one RFP at a bunch of different target sites within the HBO teaching and what you can see in blue are the results when we do not have any electroporation enhancer present and you can see that rarely it goes above 10% or maybe 20% in in this case however upon addition of the electroporation enhancer at say a concentration of 3 or 5 micromolar we see a huge increase in the editing efficiency indicating that the addition of this electroporation enhancer strongly boost the the genome editing efficiency and we recommend you using this for your own experiment so one of the things that we did was trying to find what the optimal concentration of the RMP is using electroporation with the ax maxis system so I introduced the RMP at different concentrations ranging from almost zero to about 5 micro molars of RMP and I did that without the electroporation enhancer and as she conceived for this particular site it definitely flat flat lines in that it doesn't matter what amount of RP you introduce into the cell the amount of genome editing is low again this is one of the hprt size that was in the previous figure as well if we introduced an equimolar amount of the enhancer compared to the RFP we find that especially at higher concentrations we find toxicity so when we compare that to a standard amount of 3 micro molar during every condition we found that this actually works the best where it doesn't lead to any toxicity so looking at this the addition of 3 micro molar enhancer generates up to 25% editing efficiency for this particular site in heck 293 site sorry in hex energy cells now this is a more potent site that we also tested where I did a similar experiment and here you can again see that the absence of the electroporation enhancer leads to fairly low editing efficiency however edition of the electroporation enhancer really boosts it and especially at lower concentrations of your our RMP so we would if you want to reduce the amount of RP that you use we would recommend you to use first of all the electroporation and an answer and the mic testing different concentrations you might find the concentration that works works best for you again here we find that addition of an equimolar amount of the enhancer leads to some toxicity in the cells so again we recommend just use three micro molar enhancer so these this is the same site as I showed over here Hector 9 3 the but instead of using the maxilla the effector system I also looked at editing efficiencies that we can achieve using the neon transmission system and over here you can see that the benefit of the enhancer is slightly less prevalent still it works especially at self optimal conditions and again the use of an equimolar amount of enhancer leads to some toxicity so we would recommend to use in this case one point eight micro molar of the enhancer very my here we go so here I compare the maxis system in grey the nuclear faction system with the neon system where I looked at editing efficiencies using these optimal conditions of 5 micro molar RRP for the nuclear faction system 3 micro molar and enhancer for the neon system 5 micro molar RMP and 1.8 micro molar and an enhancer and we tested a couple of different size on the HP RT gene and you can see that the editing efficiencies can be around 50 to 60 percent and for some sites that are maybe less potent can can drop down somewhat but we can see that the addition of the enhancer increases editing efficiency and it it works in both systems now one of the things that you might wonder about is whether the electroporation and hazard can integrate into your cup site and if that's something that you don't want which I can imagine we did do some testing using NGS or next-generation sequencing to look specifically at the indle profiles that are generated by CP of one genome editing so we tested a particular site in hack 293 cells and over here you can see the percentage of other sequences in relationship to the cut side so at 0 that that means there is no editing taken place in the absence or the presence of the electroporation enhancer as you can see the absence is in blue the present is in isn't-isn't orange and overall the in dell profile is similar between the presence and the absence of electroporation enhancer indicating that this edition of the single-stranded oligonucleotide does not alter the in dell profiles we also did not find any integration of the single-stranded oligonucleotide sequence that into the cut side indicating that you don't have to worry about that the other thing that we tested was to see how active the RFP system is so we transfected actually three cells or HeLa cells with 5 micro molars of rfp 3 micro molar enhancer using the metro system and we looked at the editing efficiency at different time points 16 24 48 and 72 hours and we saw that the editing efficiency is highest at roughly 48 hours of incubation of your RFP and this kind of was the same between hi to nine threes and HeLa cells so we recommend you if you want to look at the editing efficiency to wait 48 hours also what you can see over here is that the editing efficiencies can differ from side to side as is depicted over here and it can differ even if you have similar sites between different cell lines so it does not always translate well for instance this side 38 186 that's targeting the cell strand generally leads to editing efficiencies of about 50% in HEC 293 cells but topside at 30% at HeLa cells so there is some difference from cell to cell so if you are looking at a or targeting a particular location it might be worthwhile to test more than one side and we usually recommend the use of traits of three or more CRISPR RNAs to to test for your site I'm sorry one of the things that we tested briefly or that I will talk about briefly is HDR or homology directed repair using CPF one this is of line of resources that's very active in our in our research group now and we hope to disclose more information as it as it comes along but I briefly wanted to show that you can use CP of one for homology directed repair so for this experiment we have used a heck tonight three cell line that Stapley expressed with CP f1 and we and we introduced the CR RNA together with an HDR template the HDR template that we are using is a single-stranded oligonucleotide and it harbors an eco are are one side and that is depicted here in the other thing that it has are two homology arms that that that line both sides of the cut side so we are targeting to introduce this eco are one side into the cut side so we have flanking arm lengths of different lengths ranging from 27 to 92 nucleotide long and this is the total length of the single-stranded oligonucleotide in this case we used Ultra MERS that are high fidelity oligonucleotides generated by IDT so over here you can find the results you can see the results of the non targeted strand and the targeted strength at the different lengths of the HDR template so the blue dots over here is the editing efficiency that we observe of using this particular C or RNA and we get about 70 to 80 percent editing efficiency of the site that are successfully targeted using HDR we find that the non targeted strand over all leads to higher levels of editing efficiencies especially when you use a length of about eighty to a hundred million nucleotides long but like I stated before this is still active but it definitely works and we can get at this point integration of about 15% another thing that we looked into was the delivery of the CP of one RMP using lipo section and we use RNA I max from thermo Fisher we tested a couple of different ratios of your see RNA compared to your CP upon protein and that's depicted over here 1 2 1 2 2 1 and 5 2 1 we're over here you can see the concentration of the CP of one protein so in this case we have 30 nanyem or in this case we have 30 nano molar CP of 1 and 13 and molar of c RNA in this case 60 nano molar of C RNA and 30 nano molar of CP f1 and what you can see or what we found is that we did observe some toxicity when that ratio was was was altered at the higher concentrations of CP f1 we also surprisingly found some toxicity at low levels of CP of 1 and we don't really understand that at this point but if you want to use lye perfection we recommend using the RMP the concentration of either 30 or 50 men and molar and do equal molar amounts of your CRNA in your CT of one protein and the the last data slide you can find over here and this is something that you can find in the user guide where we have determined positive controls for the CP of one system for either human Mouse or or the red genome and the sequences are listed over here so we would strongly recommend you to use a positive control because it can because if if you're not sure whether your target site is active you at least can determine whether your system is working for the human side these are the results that we tested in hack 2 9 3 cells where you get up to 75 percent editing efficiencies for the mouse lines we have used the hyper 1 6 cell line for rat the rat 2 6 cell line and the editing efficiencies are somewhat lower but still rope or robust again this is something that you can see and find on our on our website it's a quick comparison of genome editing comparing casts 9 + c PF 1 again CP of 1 is is is particularly good for 80 rich genomes or when you're regionally interest is 80 80 rich it only has a single seer RNA and it does not and c Pavano does not use a tracer RNA the length of the CR RNA is about 41 that we recommend CP one has five prime over hangs upon coding where.is.cass nine cuts blunt and the pam sequence differs now the products that we offer you today can be found on their website we have the optimized to chemically and the length optimized CRISPR CP of one CRISPR our RNAs and we have the CP of one protein for sale and the electroporation and and an answer and you can find the sequences and a user guide it would also invite you to look at the CRISPR Cassadine system and local pricing can be found on the website as well so if you have any questions please let us know you can always talk to a person by giving us a call or through email or by or by phone and that one thank you for your attention okay a great talk Ralph and we do have a few questions already but I just want people know we've got about twenty minutes for questions if you have some questions that you'd like to ask you can type them into the GoToWebinar control panel into the questions box we'll get to as many as we can if we don't respond to you right now we will respond to afterwards by email so yeah by all means ask any questions that you have and get through as many of these as we can so you ready to go Ralph yep all right so the first question that we have is is there any difference in the editing efficiency due to the overhang that's created by CPF one being easier to rejoin than a blunt end yeah yeah that is that is that is definitely true that's one of the hypotheses that is out there and it's very likely to be the case yeah yeah also that is kind of one of the areas of research that we're actively looking into now if whether we can use these overhangs or ACR and making that more efficient okay do we have any information on whether or not this could be used for editing in plants like rice or corn or anything yeah yeah yeah there's no there's no as far as I know I think it works in these organisms so one of the things that you have to keep in mind though is that the different CP of one proteins coming from different bacterial strains can either it was definitely tested if it was active in mammalian cells or not and I think that the ones from private Ella and fenestella are also active in more plant-based regions but right now at this point we only offered the acid amino caucus version okay you showed earlier that you can use a range of lengths for the proto spacer region is there any increase in off-target effects if you use a shorter proto spacer region oh that is that is that is a that is a great question it's something that we are currently investigating - yeah and that and that requires a and non-targeted NGS approach basically to look at at the cutting efficiency on target and off target along for various sites but yeah that's where but we're looking into that okay I'm gonna ask you a couple of questions here about the electroporation enhancer so the first question is can you say a little bit more about what the electroporation enhancer is yes so the idea behind the electroporation enhancer is that it is a single-stranded oligonucleotides without homology to at least human read or mouse genomes if you have it working with a different organism we would we can find out for you whether homology exists please shoot as an email for for that the idea behind it is that the negatively charged DNA might might be beneficial for the delivery of the RMP into the cells the exact mechanism of that where where we don't know I have a hunch that it might lead so during electroporation pores are formed within the cell membrane and it is thought that the negatively charged illegal nucleotides can form a conductor or like micro conductor sites upon the cell membrane and that is where pore formation during electroporation is most likely if you have a somewhat positively charged protein like CP f1 it can it is more likely to be enclosed presence of those conduction sites and those pore formation sites so that's kind of where we think it it it works especially if we kind of take a look at the differences between lipo fection for instance where we do not see any benefit of the addition of the electroporation and/or the single-stranded oligonucleotide and that's why we refer to it as an electroporation enhancer because it specifically seems to be beneficial during electroporation of your RMP so it doesn't work with like other methods like if your transfecting a plasmid or if you're doing a chemical transaction of the RMP you know is it no no in then particular for RMP and in the setting of electroporation and the sequence also matters like the sequence of this electroporation enhancer it's different for CP f1 for example than it is for our cast 9rp yep true yes yes okay I think that covers a lot of the electroporation enhancer in questions there was a question I think this was only slide 24 when you were showing the editing efficiency over time that that graph it goes up to a certain point and then it kind of dips a little bit at 48 hours do you know why there's that dip at 48 hours so that might be because the cells that are targeted might have a slight disadvantage in growth so your unedited cells might grow somewhat faster than your edited cells so that's why we can can see that small drop that's interesting all right and that's also you know especially if you want to create particular cell lines that have specific mutations and if you want to do a dilution series to I solo to isolate monoclonal we would recommend to do that early early on because you might have a disadvantage when you're unedited self might over curl your edit itself there's some really great questions here so here's the next one and I'm sure we'll come back to this topic in the future too we have some stuff coming soon this person was asked if either cast 9 or cpf one works better if you want to do homology directed repair experiment um well when it comes to homology directed repair there's a couple of variables one of them is for instance how active your target site is because we can see both forecast 9 and for CP of 1 we can see that certain site sites cut better and then others so you need to have a potent target site then the other thing that my play role is the length of your nology arms and whether that is long enough or short enough it looks like longer is not always better but I think it also depends on your friends is Europe melt melting temperature of your region of Europe homology on so that is another variable the concentration of your RACR template is another variable that can drive it up or down and one of the things that we're also looking into is can we use the electroporation and answer together with HDR template to drive the HDR up even even more but these are all things that you have to consider when you want to do HDR also the target strengths are you are you designing HDR templates that are homologous to you non-targeted versus your targeted strengths we can we can definitely see difference with TPF one there let me think yeah on top of my actor there might be another couple but yeah so yeah which one works works better just to wrap it up I kind of want to say you know it it just depends on on on these variables that I listed and I think that giving it a shot for for either one a can can lead to very good HDR results but it's more dependent on other factors as well okay here's a question for you how do we design the guide RNAs for our CPF one research oh please go to our website it is described over there as far as I know okay so so the only thing that that that you need to put in is your proto spacer sequence in a DNA format and the ordering tool will automatically convert it into a CR RNA also to get that that proto space your sequence do we recommend a particular design tool for them the person that asked this question was asking if if we score the off target sites like the MIT tool does yes yes that is something that we're currently working on in house I would I don't know if the mit has a scoring tool or CP of one at this point there are some tools or web web tools out there you could have a look right now as I shown in the presentation just as long as you don't take a tttt pen site I think you're you're pretty you can rule out a lot of your non workings here rnase when it comes to your on target site but how the proto spatial sequence has an effect on the off target site is something that we're working hard on right I was wondering when I read that question that they were a little bit vague about the MIT tool but I sort of assumed that they were talking about the cast 9 tool there yes yes yes yes true and that's basically looking for homology across the genome so to be honest and looked at the MIT tool myself with regards to the CP of one and whether you can just introduce them purpose base or sequence to see what the score is there so yeah so are we or any of our collaborators using the CP f1 system in mouse embryos um let me think a little bit we have a large group of beta testers and I'm pretty sure that that's being used as well I mean for the Alice embryos you can either use it through micro injection and that works as far as I know just as well for CP of one as it does forecast forecasts 9 and we do have a method on our website for the CRISPR cast 9 system where they've done injections and you know that's probably a pretty good place to start from I would get I mean that RNP you know it's just a difference in the RFP components yeah okay let's see here questions are coming in pretty quickly here hmm this is an interesting line I don't know if we know the answer to this what is a typical half-life of the CPF one or caste nine are NPS in the cell so 48 to 72 hours that is the typical half-life yeah okay and that is when you introduced it as an as a protein I mean for a plasmid expression it can linger on for a very very long time and mRNA to that just lingers along and generates longer time spans for protein expression and therefore the amount of off-target effects in increases - okay I'm pretty sure the answer to this is yes but you probably have a much more detailed answer than that do we have data for the seat CPF one and cast nine compared to Kath nine efficiency the editing efficiency when well when that when the delivery methods are other than RMP I know we do for dining yep we have looked at stable cell lines and overall so cell line stably expressing either caste 9 or CPF 1 and we see that over all the editing efficiencies are a lot higher when using a stable stat line probably because the amount of CPF one is is is higher compared to RMP delivery and yes then is then the discrepancy between the two is is not as large but yeah yeah but we looked at though that okay this is probably a pretty important question to ask actually here do we have data on using the electroporation enhancer on the bio-rad electroporator no I have not tested that one yet we do have a gene fall for at in the lab but right now I focus so far on the news reflection system and by an expat and the NIA system by its erm oh so I guess yes I can I can imagine that it's beneficial yeah but unfortunately we have to test it ourselves okay let's see here okay so here's the homology directed repair question there's a lot of these are coming in pretty fast now this one you mentioned single-stranded donor is better than a plasmid as donor for homology but how much difference is there in efficiency for HDR I think that that might be site dependent again one of the things that that that the introduction of double-stranded DNA leads to is integration into the cut side independent whether HDR is taking place or not so and that is something that you might want to prevent if you're wanting to create a very specific insertion into your region of interest and that's why we recommend the use of single-stranded oligonucleotides because the chances that integration or random integration takes places are less less likely compared to a plasmid one of the things that I haven't talked about during the presentation but one of the new products that I IDT will release is the production of long single-stranded oligonucleotide that can be used for HDR up to two thousand nucleotides long and that might open up a lot of new possibilities for people to do HDR ok I want to take just a couple more questions but I want to let people know first of all if we don't get to your question and I'm not going to get to all these because there's quite a few here on there great questions we will respond to you by email and get you an answer to your question if I haven't answered it or if if I haven't asked it or if we haven't gotten to it and then the other thing is I wanted to point out that in the chat box you'll see that there is a link to both the user guide for CP f1 as well as the slide deck for this webinar which is now on our SlideShare site so you can go link to those things right now and we'll also be sending you a follow-up email with links to the recording of the webinar and the slide deck and whatnot in the next couple of days so I just want to let you guys know that that's on the way and let's see here I want to I do want to ask just a couple more of these some people are starting to kind of trickle out of here and I know people have things scheduled for 10 o'clock or well whatever time it is in your time zone but central time for us is 10 so a couple more questions here what does it mean okay this is a homology directed repair question what does it mean when you say the non-targeted strand has a higher efficiency than the targeted strand in your heck to 9-3 cells yes so since we are using single stranded oligonucleotides we designed them with having homology to either the targeting strand or the non targeting strand and that is why we refer to it because there's two possibilities right if you're using single stranded oligonucleotides in the case of using a double stranded template then you don't have that necessity but that's why we specified it okay here's another one did you try other cells such as primary cells compared to the cell lines for CPF on genome editing not yet but we're about yes we have tested the RMP system for cats 9 in primary cells and that works great yeah in fact Ralph here's a question have we done this I know we've done this for the cast 9 system have we done CPF one in T cells I have successfully electroporated CPF one into jerkies cells and that translated really well to primary T cells when we did the cast 9 RFP optimization so again since the electroporation is its most likely cargo or is is specific to to cargo but since it's both an RMP complex I I foresee that that that will translate just as well yeah okay here's another this is an interesting question one of the things about CPF one having the more eighty rich sequences available has has anyone tested that in bacteria as delivering it as an RNP we haven't we have a bacterial expert working in the lab and what I understand from from from him is that there's there's more efficient ways to do bacterial editing other than using the CRISPR system Ted is a person as far as I can go yeah please please again if I don't know the question or I'm sorry if I don't know the answer you can we we also write them down and and we can follow up with the experts that we have here yeah we've got a lot of collaborators and a lot of people in the lab working on different different systems so yeah I think at this point we're three minutes after the hour and there are still a lot of questions we've captured a lot of them I think that we'll wrap this up and everybody who still has a question here just expect an email from us fairly shortly and we'll we'll get you an answer to your questions and thanks so much for the great questions and participation in our Q&A session here this is great Ralph thanks a lot great presentation Thank You Ann and thank you all for listening

Channel: Integrated DNA Technologies

Views: 4,643

Rating: 5 out of 5

Keywords: CRISPR, CRISPR-Cpf1, genome editing, Cas9, Cpf1, Alt-R™ CRISPR-Cpf1 System

Id: i2Q4U3EF4Zs

Length: 62min 56sec (3776 seconds)

Published: Mon Feb 20 2017