A CRISPR Q&A with our experts: You ask, we answer.

A CRISPR Q&A with our experts: You ask, we answer.

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Hello. And welcome, to this integrated, DNA technologies, crisper question and answer webinar, my, name is Malcolm MacDougall, and I will be serving as moderator, for today's presentation. Before. We begin we wanted to cover a few housekeeping items, at the, bottom of your screen are multiple application, widgets you, can use all, the, widgets are resizable. And moveable so feel free to move them around to get the most out of your desktop space, you. Can expand, your slide area or maximize, it to full screen by clicking on the arrows in the top right corner if you. Have any questions, during the webcast you, can submit them through the question and answer widget, we, will try to answer these during the webcast but if a fuller answer is needed or be run out of time it will be answered later via email, please, know we do capture all questions, for, the. Best viewing experience we, recommend using a wired internet, connection, in closing. Any programs, or browser sessions, running in the background that could cause issues, webinars. Are bandwidth intensive so closing any unnecessary browser, tabs will help conserve your bandwidth a copy. Of today's slide deck and additional, help materials. Are available in the resource list we. Encourage you to download any resources, or links that you may find useful the. Panelists, for today's question-and-answer. Webinar will be dr. Garrett Riddick dr., Christopher, vocalist, Ashley. Jacobi and dr. Matthew McNeil Garrett. Is the director of product development at IDT he has been involved in high-throughput screenings, of Si RNAs, and vitro, Christopher. Is a senior staff scientist, at IDT he has managed contract, research projects, led, process, development, for CRISPR protein, purification and. Developed. Novel CRISPR, proteins, including, alt R SP hi fi cast, 9 nuclease. Ashley. Is a senior staff scientist, in the molecular genetics, research & development, group at IDT MS, Jacoby has been part of the IDT rnd, team for over 12 years, Matthew. Is a bioinformatics, senior, staff scientist, at IDT, he has focused on development of analytical and designed tools supporting, next-generation, sequencing. And CRISPR, products. Today's. Webinar, will be moderated, by dr. Elizabeth Gustafson. Wagner. Elizabeth is manager, of the scientific, application support group at IDT. She. Joined IDT as a scientific, application. Specialist, and is currently managing, the team dr., Wagner is involved, with her providing, product recommendations. Troubleshooting. Customer experiments, and providing, customer support and an experimental, design and analysis, for, a variety of applications including, CRISPR, gene editing, the. Question-and-answer session, will be 45, minutes to an hour during, which you can feel free to submit your question at any time in. Case you need to leave early or want to revisit this webinar we are recording the presentation and, will make the link to the recording, available on our website a few days after the presentation. We will also post the recorded, presentation on, our YouTube and Vimeo channels, you will receive links to these in a follow-up email also. An on-demand, version of the webcast will, be available approximately. One day after the webcast and can, be accessed using the same audience link, that was sent to you earlier so, now let, me hand it over to Elizabeth. Thank. You Malcolm for the kind introduction as a, reminder if you have a question, for our panelists, at any time during the webinar please.

Type It into the Q&A box. We. Will begin today's webinar with, a brief introduction, and review of the CRISPR technology, presented. By Chris a senior staff scientist, and our CRISPR R&D, group so. Now I'll hand it over to Chris all. Right thanks Elizabeth, I'm. Going to talk just for just a few minutes about, CRISPR. In general, and then, give, a brief overview of the different types of products that IDT, has to contribute to this space. So. In general we talked about CRISPR there are two very popular choices, for doing CRISPR. And live cells and those are cast, 9 from strep pyogenes and, cast. Well they are formally known as CPF. One from ascetic caucus species these. Are both RNA guided, DNA, and a nucleases, and they're. Fairly. Similar with, some very key differences, and probably, the most important, of which is that they're able to target very different, types of sequences, in the genome cassadine. For example target spam sequences, that are in G gee where, as CPF 1 targets, triple, TV, so you can sort, of span the gamut of of GC, content with these two enzymes. And. Regardless of what enzyme we're talking about people are using. Them to generally do one of two things in living cells on the bottom left-hand side people. Are generally. Trying to. Allow. The native, repair process, to join ends from a double-stranded, break and, in general this is an error-prone process so. You typically, get an insertion or a deletion, of a couple nucleotides. This. Occurs to the non-homologous end joining. Pathway. And if, this is for example in the middle of an or if you can imagine this is a simple, and easy way of introducing, a knockout. Alternatively. On the, bottom right-hand side people, are using these enzymes, to introduce a double-stranded, break and then to introduce. Into that cut site. Virtually. Any DNA sequence they want by virtue of putting homologous, arms. Or ends on a template. DNA sequence, and. Allowing the HDR. Native. Repair machinery to introduce, that DNA add. The cut side. And. You can introduce CRISPR, reagents, by a wide variety of mechanisms into. Cells you. Can purify, the capsule, and receive the f1 proteins, use chemically, synthesized, RNA you. Can also supply, these reagents, off of, DNA templates, but in the form of a stable. Expression, cell line you can amuse your transcribed capsule enters TP upon mRNA, and, alternatively you can express both the protein and the guide, RNAs off of a plasma door or a lentiviral. Expression, vector. At. IDT we promote the use of our MP or ribonucleoprotein. Delivery for. Very few important, reasons. The. First is wood of which is we. Want to avoid unwanted. Stimulation. Of the immune system any problems, with toxicity and cell death and, we have found that the RMP delivery, gives us the. Lowest amount of immune. System stimulation, and second. Secondly. We, want to reduce the opportunity, for unwarranted, off target editing so casts 9 as. I'll show in a bit. Has a propensity to cleave off target and we want to avoid that at all costs and what we have found is the longer, these reagents are in the cell you have the most opportunity, for doing off target editing so so we want to use the delivery format, that allows. Their, Bri agents to do it they're supposed to do and get out an RNP does that very well. So. Moving right along I'll talk about what. I DT, has to contribute to this space and the various types of RNAs, and proteins we have. And. I'll start with the guide RNAs we. Sell three different forms of cast nine guide RNA the, first two forms and the the left and middle got. A standard two part where you've got a universal, tracer RNA that's heavily modified and. A targeting, crispr, RNA or, CRNA, that. Is minimally, modified, for the standard two part system and, more heavily modified for the xt system as we call it and.

For. The vast majority of users who are either working in, cast, iron stable expression, lines or doing, RMP, delivery, and the majority of cell types the standard two-part system is very, efficient. Editing. But. For people that are working in, either nucleus. Or in cell lines challenging, conditions or, alternatively, introducing. These reagents, Co. Delivery, of the chemically, synthesized, guides with, either cast and expressed no plasma or mRNA, a more, modified, rnase, needed so that they can persist long enough for reasonable. Expression, of caste 9 and, in. Particular for, Co delivery, type experiments, those. Might be cases where you need them more expensive sgrna, option, and we certainly offer that as well with standard. 40s modifications. Importantly. All of these options have, a very fast three, to five day turnaround time. So. Which. CRISPR, n'oubliez should. You use and in fact there are a variety of options we have options. For both cast line and cast welfare cpf one and. The approach we've taken is to identify the, biggest, challenges, with these enzymes, and come up with proprietary, solutions, to those problems and. For wild-type cast iron we found as I mentioned before this. Has the highest propensity for unwanted, off target editing, so. We employed. A bacterial. Screen to find. A more space vikas nine very and in fact we found, one in productized, it called it the altar - cast nine, and. We find that this enzyme maintains, the vast majority of on-target editing while simultaneously, reducing, off targeting, and, then. Forecast over CPF 1 we, find that the native enzyme has, relatively. Low overall, enzymatic. Activity, in live cells so, again we screened in a bacterial, system to, find one that has enhanced overall, activity in this, product which we are going to call the altar caste 12a ultra. Protein is not, yet available, for, for spa sale but, we expect it to be on the website in the coming months so. Moving, on to repair, we have a variety of different options for doing, HDR. With. Your CRISPR experiment, we offer templates, such as a short. Single strain at all agos ultra MERS up to 200 bases it can be used for this process, we. Have much longer enzymatic. Lee derived mega more single-stranded, DNA fragments. For, producing gene length fragments, and then. We. Also have double-stranded. DNA fragments, in the form of G blocks or custom, genes that are cloned on a plasma for, those, that can use double stranded, HDR. Templates in their system and any. Of these options can be used for both cast line and CPF, for our cast web a. Coming. Later this year we're very excited to announce an HDR template. Design tool and, this was developed internally with a lot of empirically. Generated. Data, it's. Going to help you with designs, in terms of how long your homology, arm should be what, the distance should be from the available Pam site it's. Going to help you. Whether. You're trying to with, silent mutations, it's going to help you with the appropriate although modifications. Etc, and it will support designs, up to two kilobases in length, and. Last but not least we have available now on the website a small, chemical, molecule an HDR enhancer. That. In a variety of cell types has the ability to dramatically, increase the rate of HDR. And. Finally, we have a couple different options for, the detection of genome editing events, after after. Using the CRISPR reagents. First of which is a less. Expensive and, and faster, approach using, PCR, and the t71, mismatch. Detection, system so. Essentially you're amplifying the, region that, encompasses.

Your CRISPR, cleavage event and. Taking. Everything in the pool and annealing, it and any mismatches, will represent a successful, editing. Event the product we have in this space is the altered genome, editing, detection. Kit we. Find that this is a relatively good estimate, of both on and off target editing events very fast easy and cost effective but. It can struggle to give you very, accurate quantitation. Of editing at certain, sites or at sites where the editing is greater than 50 or 60 percent. So. For those customers. We recommend, moving on to an NGS, sequencing, based approach and, we have a very new product in this space targeted, amplicon, sequencing, approach we call ramp seek, this. Allows you to have. Submit. Specific. Genomic sequences, to IDT, and will design amplicons. For, however, many on and off target sites you have, upwards. To the thousands, at one time these are all amplified, in a single pool and. Sequence allows, you to quantify the, percent target editing at all these sites and also gives you a picture of the different types of repair outcomes that happen at all of these sites helps. You to save time and resources if you have a lot of sites to look at at. One time. I'm. So to just sort of summarize this is what, we call a complete solution, from. The beginning, design to the final analysis, of CRISPR editing events one thing I didn't talk about in our design is the Cassadine. Guide RNA design tool it will allow you to find pre-designed, guides or customize. Your own and do design checking. Again. Just to reiterate will have an HDR template, design tool coming very very soon, to. Do the cutting we have this very. Several. Different options for guide RNAs for both Cassadine and Casco Bay we. Have a few different options for CRISPR proteins whether you're using other of those enzymes. We. Sell for the repair pathway. Both, ultra Murs of a single strand DNA fragments. Very long mega Murs and, we have the Ultra HD or enhancer and finally we have two different options for doing both fast and more rigorous. Genome editing detection. So. If. You have any. More questions, you can for those on to application, support at IDT DNA comm at anytime and, they'll. Either answer your question on the spot or direct you to whichever. Scientist, is best fit to answer that question, so. With that I'll open it up to the Q&A, session. Great. Thank you very much Chris for that informative introduction. With. That foundation, established will, now begin to address some of the questions that have started to come in from our webinar, participants, so. For our first question you. Mentioned, Ramsey, custom, panels, - as a CRISPR, on-and-off target, activity. What. Does the normal turnaround, time to. Get a target panel how. Should i generate, my target, list and how should I analyze, the NGS, data, so.

To Get. It. The. Way our process works is that we request. The use of you, initially submit your request to our applications. 214 from. There once the request. Makes it on to our design team it's typically within a couple of weeks that you'll get a design back and they'll, work with you directly to get a design that fits your needs. And. Elizabeth. What was the second part of your question. I. Pull it up here. What. Is the normal turnaround, time and how should I generate, my target, yeah so half right so generating, the target list can be a complex. Process with CRISPR cast nine cast. Nine we know can. Act at both on and off target sites you. Can use a, number of different methods to identify those, off target sites you. Can use bioinformatics. Tools, which are available on the web and there are many examples that are out there we. Find that these tools tend, to be very comprehensive when, it comes to identifying all possible, off target sites however. Their specificity, for, identifying, which ones are most likely to show up in your set isn't, always a. Reasonably. Sized list, instead. We recommend using empirical data to generate. That target list using tools such as guide seek site seek or circle seek within. Our Rd group, to use a tool called guide, seek from, shamed our side, this. Tool is it. Has both. Recommended. Web bench reagents, as well as analytical, are an analytical, tool for analyzing the NGS data as an. Output from this tool you will generate a list of genomic. Coordinates that, are identified. At which we can confirm. There are off target, sites that. List of off target sites can, be fed into our design, engine and we can design a wrap seek panel that, will. In that will generate amplicons, for all of those targets up to a total, of 5000 possible, targets. All. Right it's great thanks Matt for. Our next question. The. Question is do we have a GMP. Option. For nucleases. Chris. Could you address that yeah. So so we have um we sell both research grade wild type and high fidelity caps nine variants, but. Just recently our commercial, partner, Aldebaran, has. Released GMP. Versions, clinical, grade versions, of, the wild type Caston, as well as a licensed. IDT. Hi-fi which they are calling the spy-fi GMP, nuclease so so, yes they are available.

Through This commercial, partner and you can get more information on the acquiring both at. WWL. Devran, com. All, right great so for our next question. The. Question is hi I'm an undergrad, at UMD, currently, doing research with, AB de mer selection, I've been, interested in gene editing and I'd like to ask what kind of subjects, I should study to get the context, needed, to move to learning, CRISPR, protocols, Garret, you want to take that one yeah, sure, that's. A good question or an interesting question. The. You. Know really was true, about all, of what we're trying to understand, with regards to CRISPR editing and I. Already talked at a conference one. Time that, suggested. That this was a case that you, know there the. CRISPR. Caste, machinery, is nothing more than a fancy nuclease, and, everything, that happens downstream of that inside of a cell is DNA. Repair and understanding. The DNA repair, mechanisms. You. Know Chris broadly, identified, those. Because. It's frankly easier to talk about in terms of two funnels non-homologous. End joining in homology directed repair. However. What. Is also true about that is there are several other. Subcategories. Of. Non-homologous. End joining micro, homology, mediated and joint etc. That fall under these two larger umbrellas, so, if. I had to recommend to. Someone what to study in in greater detail, it would be some, sort of cell biology based course, that delves. Into those, DNA repair mechanisms, that, that's really essential knowledge in. This field. Alright. Thanks Garrett. So. For the next question, the. Participant, is asking, is there a method to analyze unknown. CRISPR. Based modifications. And. I'm wondering if potentially. This. Participant, could clarify, their question, a little bit to. Maybe. Shed. Some light on what they mean by unknown, CRISPR, based modifications. That might help us provide, a more, informative, answer, for you, so. While we wait, for that clarification we'll, move on to the next question and then circle back to that one I could. Speculate, on, that why not, so. I this, may not be what the person, had intended but there are things that we don't really understand, that well that I don't believe, there are good options to evaluate, at this time and one, of those would be things like, unintended. Genome, translocations. And things that happen that are not just simply on an off target, editing more complicated, things downstream, and there. Are, academic. Or published. Techniques. For identifying the spectrum of things have happened in the whole strain of breaks but I don't think any of those things are easy and unfortunately, we don't have a commercial you. Know product of any kind that addresses, that but if that's what was being asked I think that's a great question. Arnie. Great thanks Chris. So. For our next question I push this how can I estimate the, amount of edit, obtained. Using the altar crisper cast nine or, cpf, one systems. Mad, or Garrett was one of you want to take a stab, at that one yeah, sure I can talk about the, first level I'm estimating and what the entity would be as Chris alluded to again back to the introductory. Slides the t71, endonuclease. Method. Is, a great way. To probably. Quickly and pre. Easily, assess, and again. At the estimate. Level what the editing efficiency, is to. Get more into specifically, the, quantification of, what the editing is and in a, population, of cells how, many what, is a percentage of the alleles, that are indeed edited, or that. Remain wild-type. Matic. Spoke to this earlier but, this would fall under the realm of our ramp, seek product where, we could use. Amplicon. Based sequencing in a, multiplex, fashion. Reliant. Upon the RNA. CH cleavable. Primers, that that allow us to again multiplex, up into the thousands, of targets. Amplified, at once. This. Not only at the on target site but as well as off target sites gives, us the ability to. You. Know very precisely quantify. The amount of editing that is going on at the and the, known targeted. Sites as well as any putative. Off. Target, sites as well. Great. Thanks Garrett for. Our next question its how should I store the cast nine and cpf one proteins, and how, long are they stable, for, Ashley. Would you be able to answer that one, yeah. So. The. IDT. Cast. Nine and cpf on proteins, are very stable proteins, and we've generated. Empirical, data showing, that these, proteins can be stored at minus, eighty, and minus twenty out to two, years without any loss and activity, we've. Also done additional, short-term.

Studies To see if these, are accidentally, left out at room temperature anything. Like that if there's, any immediate loss in activity, and we've. Seen actually there them, at full activity, maintained, if these are left out for several weeks at room temp we, wouldn't suggest stirring them that way we would suggest minus 20 and minus 80 but. These, are very robust. And stable proteins. Okay. Great thanks Ashley. So. For our next question, we. Had great success, with knockout. System, but, had a hard time for. HDR, snip, editing, using, single, stranded all ghosts do, you have recommendations. For optimization. Ashley. Would you like to address that one sure, yeah that's it that's a big area of research or a big area of focus our research group is actually doing. A lot of work on to optimize, the. Modifications. The, arm links and such. To make your HDR as efficient as possible later. This year actually we will have a design, tool that will help incorporate, all of that but, now some. Options are the we have an HDR enhancer, which Chris, mentioned which is a small molecule compound. That. You can add to your your. Media and have. Your. Cells incubate, in this for 12. To 24 hours, during. After delivery of your CRISPR components, and this can actually and some. Type some, cell types increase, HDR, rates up. To six fold we've seen in some, cases and. So, well is the, modifying, your ultimate, template so it's been published that. Adding. To phosphoryl, thio eight linkages, to the end of. Two. To each of the 5-prime and 3-prime at end of your altmer of your single strand all ago is going to increase, HDR, rates and that is true versus, using an unmodified DNA, template we've, also gone further to identify, even additional. Modifications, that further boost this. Editing. The. Other thing that you can do is to add, in synonymous. Snips that will, reduce. The ability. Of cast nine to recut, your target site generally. The rule of thumb is that you want to add in a couple of sniffs particularly, toward the, pam site itself if you can change.

Those Gg's to some other nucleotides, without. Changing, the coding sequence or otherwise. Affecting. Your desire, outcome, you'll. Get an increase, in editing, efficiency. One. Thing I would add to this is you know if you're getting good. Knockout efficiency, but HDR, is not working. Well you, know it's hard to know what could be the. Holdup with that considering, this could be anything from plants, to animals and we just don't have more information but I would say I'm, along the lines of the HDR template, if possible. Try to figure. Out how long lived, whatever, HDR, substrate, you're using is and whatever cell line you're using and find out if that's really the choke point if it's getting rapidly, degraded. Considering. Something like a more highly modified, template. Has Ashlee suggested, that. Could be really what's what's holding the whole thing up yeah. And one, final suggest, or one final, suggestion for me perhaps. Would. Be we've. Also seen that it's some target, sites that if you're that. Generating. Your SSO nhien against, the temp the targeted, strand or the non targeted, strand that, sometimes. One will work better than the other and so, we generally recommend choosing, or that trying both because, we don't have a method offhand, to be able to tell you which one will be better, yeah. And, one, last thing is I just wanted to point out that we do have a decoded. Article on our website that does highlight some of the, optimizations. And benefits, that you can add into your HDR, experiments, to, try out these things we've suggested, here. All. Right great thanks to all of you so. We have a follow, up or clarification. Question, stemming. From the. Previous panels from previous participant, question is there a method to analyze unknown. CRISPR, base modifications and, the. Follow up clarification. Is if, one, is trying to detect if an unknown sample has, been modified, using CRISPR is there. An available, method, besides, whole, genome, sequencing. Garrett. Would you mind addressing that one yeah sure. Matt. Alluded to this earlier when he mentioned, nah I think it's more clear what was being asked here that we. Utilize the guide seek method as an unbiased. Methodology. To in, cell. Live cells. Generate. Those edits both on and off target, capture. Them with a double-stranded, tag as is published in this 2015, publication. And then, use that tag as a handle, for amplification, from. That from those experiments, from. The definition, of those validated. Sites throughout the genome so, again this is a targeted, and richmond strategy. That. That is absent. The need to do whole genome sequencing. We would then use that to generate, a list of coordinates for. Which we would design, amplicons. For, another round in a more specific round, of targeted, based amplification. Using the rapeseed technology. You. Know anecdotally we see as good. As 20%. Of reads, from a guide seek, experiment. That, are specific, for the target oftentimes it's less and that this guide dependent. And there are the factors that can influence that as well but. From, then, translating.

That Or comparing that against a ram stick experiment, often. We see greater than 95, and an office 99%. Of all, reads that are, amplifying. Or that are attributed, to a target, within the pool so you you're, able to use. The. Unbiased route, for, discovery, and then, using, the ramp state methodology, for, more. Specific, quantification, of your targets of interest I. Think. Require, a. Reference. Genome for yeah, I wonder. If that was the question unknown sample, meaning that they don't even know, there's. A rough because I know there are some of these methods they don't require reference, genome but if it's bliss, or bless yeah, in that case you'd still want to do a comparison, against. Something that could become your reference and untreated and, untreated that's right yeah. And in your if. You go that direction, you. Might still have to do at least once, something the equivalent, of. Whole. Genome sequencing, just to go to create your background, to. Show what it could be depleted from that. Great. Thank you so we have another question that's come in related. To HDR. The. Question is please can you shed more light on the mechanism. Of HDR. Is the single-stranded donor template, inserted. Into the cutting site or, is it copied, to repair, the damaged DNA mat, perhaps you could address that for us sure so, one of the the real, advantages, of using the homology, directed repair. Pathway. Specifically. With a single-stranded. Template, is that, it won't, go in as it. Won't just be inserted, into the target site itself if you use a double-stranded. Template, there is the possibility that it can go in double. Blunt into that target and, it can go in, and. Fractional, increments, as well and. This is more we typically. Describe it as going in through the nhej a pathway so. If you go in at. Half x or up to two x of your, fractional. Length with. The with. The homology, directed repair pathway. It will be copied, into the site using, those homology, arms on either side of your desired mutation, to, identify, where in the genome it should, be where. The cell needs to find. Its match and use. This the new desired, sequence to, generate, the repair because. You're copying from, the single-stranded, template, you're, able, to introduce. Small. Changes, from the original desired. Sequence into. The genome such as an insertion or or. A desired, deletion. All. Right great thanks Matt for our. Next question how many cycles of design, CRISPR. Analysis. And redesign, based. On targeting, results, should, one anticipate, is one. Cycle typically, sufficient or, should one plan for many cycles, Ashley. Would you be able to answer. That one, yeah. So, so. I think my cycles your meaning. How. Often is it going to how, many how much of optimization. And the design of the guide RNA goes.

Into This and does, it often take many many, tests to get that to happen on so. We've. Really worked hard to develop tools, to make. This very efficient, to begin with so, at IDT we have a design, tool where. We've used empirical, data to. Predict, guide. RNAs that are gonna have high on target activity as well as with, the design of our guide rnase and reducing chemical. Modifications. That are we going to make these more stable thus, more efficient, in your system as well as delivering these with a cast nine protein that's all going to give you the the best chance of having really high editing. And. Chris. Burke has nine as well as cast 12a now with our innovative, new. Cast, web cast, of all day ultra doux have very high efficiency, and, these these, work very very well and so we often recommend. If you're looking at a new target work that you're just wanting to do and knock out in for example to to, look at two. To three guide rnas and as long as you've optimized, your delivery, of your cell type and with, the controls and we, do offer control, kids that are validated, guide RNA so if you're seeing high activity with those and you have that system working it. Will. Not take many rounds at all likely. One maybe two to find an efficient, guide RNA I think. It just depends on what you dean, is the success, of an experiment concerned, about off Tarija editing on targeting. Is. This a really challenging cell, type or is this you know an immortalized, cell line. Yeah. I mean if, your goal is to minimize that and expense isn't really not an option. You, know hi-fi cast nine with an sgrna. And. I would imagine and the majority, of cell types is going to give you high efficiency, and low off targeting the majority of the time yep. Yeah. I think I think that the, summary is that this is a very high a very, efficient system and depending, on what your specific application is, there are tools to really make that work well, for you. Okay. Thank you so. For our next question does. IDT, design, and offer libraries.

For, Crisper editing. Actually. You'd be willing to take that one yeah, yeah, so on as. Chris mentioned at the beginning we, do have three. Different types of guide RNA, forms. That we offer so we've got the dual guide RNA system, as well, as the single guide RNA system and if you're doing screening. The dual, system does work very well because the. Target, specific, sequence, is only 36, bases and this can actually be made in high throughput and a very cost-effective manner so yes, IDT, does, make. Libraries using. The CRNA, specifically, and then you order your trace RNA and large. Quantity, and. We've. Done work with groups, where they have done the the guide RNA design themselves we also have. Done the work we've got right now we've got stocked, libraries. For. Whole. Genome for. Kinase. For epigenome, and you know that's something I would encourage you to reach out and talk to our app support, about but that is something, that we do and can, help. Out quite a bit with great. Thanks Ashley so. For our next question are. There any are, any of the older CRISPR, system offerings. From IDT, applicable. To a prokaryotic. System, like, bacteria. Chris. Would you be able to take that one yeah. So you know the short answer to that is no we don't currently support. Editing. In prokaryotic, organisms. But. I think obviously people are doing that especially nikohl, I and the literature and oftentimes they're expressing, s. TRNAs, off plasmids, but. As far as getting you, know RMP reagents, to work and things like that I don't think there are presently good options. You. Know there are always things. We do in bacteria, to engineer nucleases, that require some, of this type of work so you, know there. Could be some follow up from this if somebody wanted to write into applications, support, of that in T DNA comm and we can funnel. That question, to the appropriate, people. All. Right great thanks precious, our. Next question. Is also another follow-up related, to HDR, and. The question is is it better using. Single, or double strand, DNA for, HDR repair, when. Using double strand DNA would, it be better using, PCR, fragment or, plasmid. DNA and, what. Is the best size for the, arms, thank. You, Matt. Would you be able to take that one sure. So, we strongly encourage people, to use single stranded DNA the. Risk, with double stranded DNA is, that the entire, fragment, can be introduced, into the target cut into the target cut site through, the non colleges and joining pathway instead of homology, directed repair, and. When that happens, you can get the homology, arms inserted, into the cut into the cut site as well and. This is oftentimes not desirable, and can lead to things like frame shifts and. Other undesirable. Effects. With. Regard to homology, arms the, for single-stranded, Allah goes we have, studied this quite a bit and have found that the amount. Of inserted, material, if you want to introduce an insertion. Really. Changes, what we would recommend in terms of the arm length for, short insertions, and these are ones that I would I would, characterize where the entire all ago could. Be down would, be under or we would be within our ultra length which is two hundred bases or fewer, then. We would typically recommend using about forty bases of homology that, are that. Are equally, sized on both strengths, sides of your desired mutation, for, longer, insertions, where. You, want to introduce, say. Something. The size of GFP, so something much longer you might want to go out to 100, to 200, bases I'm all 200. Base homology, arms on either side for. PCR. Fragments, if you're gonna use PCR, then you're going to be generating a double-stranded.

Molecule That, runs, in those same kinds of risks that I mentioned before and the same goes for plasmids. All, right great thanks Matt. So. For our next question, and. The. Participant, writes it may be a stupid question there, are no stupid questions. But, I wonder if the length of the, G RNA has any effect, on targeting. Success. Ashley. Would you be able to answer that one for us I, sure. So. There. Has been some, work and. Literature. Out there of changing, the length of the proto spacer, sequence which, is, canonically. 20, bases for cast 9 and. Shortening, that has, been published, to increase pacifist, or taneema to say 17. Or 18 bases. We have found though at many sites when you do shorten, the, length of the protis baster sequence specifically, you do also take a hit, in on target activity we. Typically for cast knives suggest, using 19, to 20 bases for the proto spacer sequence if. You, referring to the full guide RNA sequence. IDT, has done. Where. You're now think talking about the conserved, region, IDT has done length optimization, studies to to. Truncate that down to where. You still maintain activity. But if you do truncate, that down, too far it will become inactive, yes, if, I, address the question, correctly. Great. Thanks Ashley, the. Next question is what are some of the challenges for, RMP, delivery, to plant cells I know this is a common question Chris. Could you potentially speak. To some of those challenges, yeah. Yeah this is really a tough one there actually have a lot of challenges I would say one. Of which is, simply. The fact that a lot of the times you're, working under temperatures, that may be not ideal, for. Cast, 9 or. Depending, on which variant, you're using casts 12 a and. Another. One would simply be the genome GC. Content so, there may not be a lot of cast 9 ngg, pam, sites available in, whatever, plant species you're working in because plants are inherently very 18 rich so. For, that reason I think a lot of people have gone to use cash flowing because and in particular the lb, or lactose. For AC variant. Of cash, flow away because it works at low temperature, whereas, the AAS or a cinnamon dukakis variant has, been reported to not work that well at. That temperature, so. I think finding the appropriate target getting. The existing, options to work well, has. Been sort of a challenge but I think one, of the products, as we mentioned that's going to be coming on the market very soon is our, AAS, derived, cast, 12a ultra, protein, which. Internally, we've shown works. Very well at low temperature, so. This may, be something of interest to a wide variety of the plant, community. Another. Thing I would point out is the sheer size or. Of plant. Genomes relative, to other. Species, are very very large where specificity. Might be something. Of an issue particularly. For something like wild-type cast nine so. So for those people that are concerned about specificity, implants, I would say obviously, our NP is the right option if, it works in your, particular. Plant. Cell line or plant, but. Something like a hi-fi cast, nine might be an option or we know that casts twelve a is, inherently, very high specificity. Thanks. Guys, our. Next, question is again about ramp seek is the ramp seek system, for crisper library, prep method for, Illumina, NGS, sequencing, if not, what, library, prep method, do, you recommend. Garrett. Could you answer that one for us sure. Thing, indeed. The ramp seek methodology, not. Just for CRISPR systems but in this case we're talking about CRISPR, it is for the Illumina platform, we. Have seen, results and. You. Know mostly from the my. Seek but also next week I. Would. Also add that in, the in the design we're typically using, to. Buy 150 sequencing, in the desire the amplicons is in the array insert. Size in the range of 120. Base pair up to twitter base pair which. Works nicely with, to, buy 150 sequencing, as well as our data. Analysis, platform but yes indeed the, final libraries will be prepped. And ready to go for a little bit of sequencing. Great. Thanks Garrett so. For our final question today that questions, a two-part, question first. Would deep learning health, the CRISPR off targets, and. Secondly. Could it be possible to determine in, Dell sequencing. After. CRISPR Cassadine, somebody. Answered them in the opposite order in which they were asked so. First, off is it possible that to, analyze, the in Dell profile, and the answer is absolutely yes, so. Using the ramp sink technology, that that, Garrett highlighted, before and we've talked about a few times over the course of today's QA. We. We're. To get Illumina, sequencing, of the. Targeted, regions by, for CRISPR and both on and off target sites using.

Sophisticated. Analytical. Pipelines. Some. Of which are available online, such, as the Christopher s O'Toole from Luca penelo you're. Able to pull, a load or tease out the in the specific in Dells that result from Chris. Berg cast 9 or seek a, squall they were paired, as. They, were paired as they are repaired, by the sell those. In Bell's can, then be fed into a number of predictive, models, others. Have and there have been predictive. Models that have been generated. Using a, number, of different approaches, anywhere from position-specific. Scoring. Matrices. Or PSS. M22. Random, forest methods to neural and even in a rare case using. Neural networks. So. Yes there are methodologies. Where, people have have, used or attempted to use deep learning methods to predict, CRISPR off target sites. What. I would say is that these these methods have have. Gradually improved our understanding of what. Sites are likely to be targeted off, target, with CRISPR cast 9 but they. One. Of the limitations, of deep learning methods, is the sheer amount of data that you require to, to. To, use them adequately, and. With. More data will, be more, able to use those deep learning methods to further enhance our, ability, to predict, where, CRISPR will land off target and edit. Alright. Great thank you very much Matt so. With that final question will conclude today's CRISPR, question, and answer session, thank. You all for sending, in your thoughtful, questions for discussion I'm, now. Going to turn things back over to Malcolm to conclude, our webinar today. Thank. You Elizabeth, I want, to thank all of you for attending today's, webinar I would also like to thank Garret Christopher Ashley. And Matthew for being part of our panel as well, as Elizabeth, for conducting the question and answer session this. Is one of a series of webinars we will be, presenting, on CRISPR as well as other top we, will email you about these future webinars, as they are scheduled, also, as a reminder a recording. Of this webinar will, be posted shortly, on our website and at WWE. To, become, I, DT D na VI o there. You will find several other educational. Webinars, on such topics as next generation sequencing. Genotyping. QPCR. CRISPR, and general, molecular, biology, thank you again for attending and we wish you the best of success in your research.

2019-03-28 23:55

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