Increasing genome editing efficiency and specificity with optimized CRISPR-Cas9 guide RNAs
And welcome to this integrated, DNA technologies, webinar, increasing. Genome editing efficiency. And specificity. With, optimized, CRISPR, cast nine guide. RNAs my. Name is Sean McColl and I will be serving as moderator, for, today's presentation. Today's. Presentation, will be given by Ashley Jacobi Ashley. Is a senior staff scientist, in molecular genetics research and development, group at IDT. Ashley. Has been with IDT for 12 years and during, that time she has been an author on 18 manuscripts, published. In peer-reviewed journals, contributed. To numerous patent, applications. And is, presented, in a wide variety of international biomedical. Research conferences, her. Current research initiatives. Focus on the development, of novel crispr. RNA sequences. And modification. Patterns that allow for more efficient, and specific, cleavage by the ESPY cast nine and AAS, cast 12a CRISPR. Nucleuses. Ashley's. Presentation, should last about 30 minutes and following. The presentation, she will answer as many questions as, possible from attendees, the. Question-and-answer session will be conducted, by Molly Schubert, research. Scientists, in the CRISPR group here, at IDT as, attendees. You have been muted but we encourage you to ask questions or, make comments at any time during. Or after the presentation, by entering your question, in the question and answers box. Also. Please note that you can expand the presentation, slide window for. Easier viewing, 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 so now. Let me hand it over to Ashley for. Her presentation. Thanks. Sean and good morning everyone today. I am going to talk to you about the different options for introducing, the CRISPR cast nine guide RNA and how to get the highest efficiency. And specificity. For your systems, and, I'll. Start by giving a basic overview of the genome editing and, then. I will spend a lot of time focusing. The different forms of guide RNAs, specifically. In. Vitro transcribed, single, guide RNAs, chemically. Synthesized, two-part. Guide RNAs which are comprised of a CRNA, and tracer RNA, that are annealed, together. Chemically. Synthesized single guide RNAs I will. Also discuss the benefit, of introducing. Chemical, modifications. Into your guide RNA and give. You a roadmap for when to use which form of guide RNA I will. Also spend a bit of time discussing, off-target. Effects and if, there are any differences, associated. With the form of guide RNA, or cast 9 used and, lastly. I will briefly introduce our, new assault our CRISPR reagent. As. I mentioned I will give a background on CRISPR, genome editing I. Will. Discuss developments. And improvements to, guide RNAs specifically. Focusing, on in vitro transcribed, single guide RNAs, chemically. Synthesized, 2, part guide RNAs where you. Have two RNA molecules, of C RNA and a trace RNA that are fused together and a. Chemically synthesized, single guide RNA I will. Also discuss the benefits of introducing, chemical modifications, and provide. Guidelines for when to use which, guide RNA form with, which source of caste 9 they'll. Also spend, a bit of time discussing, off target, effects and how, these play a role with the different guide RNAs or, caste 9 that you are using and. Like I mentioned I will give a brief introduction, to our newest CRISPR. Reagent. The. To. Comment. Most common RNA guided endonucleases. That are used to edit genomes, and living cells are cast, 9 and cast 12a otherwise. Known as CPF, one now. On the left in this slide is the caste, 9 system where. The guide RNA that. Associates, with caste 9 is natively. Comprised, of two RNA molecules, in, green. The, C RNA which. Contains the target specific, region shown, what's a thicker bar typically. About 20 nucleotides in, length.
Anneals. To the Teresa RNA in, orange which is the Universal sequence, that associates, with. The CRNA, the. Active complex is then, guided to this target specific, area in the genome upstream. Of the pam site which is an ng G and a, blunt double-stranded. Break is made now. Cast 12a on the right side of the screen. Only. Has a single short C RNA also. Shown in green which. Contains. A 21, to 24, target, specific, regions shown in the thicker bar is also. Guided to the area in the genome, now downstream. Of the pam site which, is a TTT V and in, this case a staggered, double-stranded, break is made. Today. I will focus on the CRISPR cast 9 system. To. Implement CRISPR, cast 9 genome editing you, need to introduce cast. 9 and the, guide RNA to your system. There. Are many ways to introduce these components, into your system, now. Starting, on the left in the upper corner. Cast. 9 protein. You. Can purify, or purchase cast 9 protein, 2 delivered, directly. Now. Moving down you, can also make, or acquire, a cast 9 cell line where, cast named protein is expressed all of the time. You. Can purchase or Express cast 9 mRNA, that can be transfected, directly, into your cells as well. And. You. Can also deliver a calf sign expression, cassette that will be transcribed and translated in, the cell now. Very similar story for introducing the RNAs moving. Over to the right side of the screen in the top right corner you can, deliver chemically, synthesized guide RNAs which have the advantage of adding chemical, modifications. These. Can be synthesized, as I mentioned, in a two-part form where, the C RNA and tracer RNA are annealed together or. Can be synthesized, as a single guide RNA where, the two components, are fused, with a linker loop. You. Can also in vitro transcribe. The single a single guide RNA from, a DNA template, and then. Deliver this, or you. Can deliver a DNA, expression cassette, and the linear DNA as a linear DNA fragment, or as a plasmid, and of. Course you could also combine the caste 9 and guide RNA into one large plasmid, and deliver that at. IDT. We, favored direct delivery of the ribonucleoprotein. Complex where. We introduced. Caste, 9 protein, we. Incubate cassadine protein, to send a synthetic guide rnas and a test tube and then directly, deliver that preformed, complex. One. Reason we favor delivering, the caste 9 and the guide RNA as an, RNP complex, is because, it allows for, very fast and efficient delivery. Now. I'd like to go into more detail on how easy it is to form the rmp complex. First. If you are using a two-part system you. Have the cost-effective advantage, of just synthesizing, the short RNA, component, the see RNA every, time you're looking at a new target as the tracer. RNA is universal. But. You do need to anneal these two strands together, we. Do this simply in step 1 by. Adding them in equal molar amounts, heating. To 95, degrees for 5 minutes and then, slowly cooling to room temperature for about 10 minutes and then in, step 2 we incubate the active guide RNA complex, with, purified cast me caste 9 protein, at at.
Least A one-to-one molar, ratio, for. About 10 to 20 minutes and then, we are ready to directly deliver the, RMP complex, into your system. We. Have many protocols on our website for how to deliver the RMP complex, we have detailed. Protocols, showing. How to do this via lipo, section electroporation. And we also have many, user provided protocols, for. Microinjection. Or, more niche systems. The. RMP complex, can also easily be generated, using a chemically synthesized, or in vitro transcribed single guide RNA or the C RNA and treesa RNA are already fused together the. Method is the same however, there was no need to pre anneal the C RNA and trace earn it together as they have been synthesised together otherwise. You incubate, your single guide RNA with your caste name protein for ten to twenty minutes and you're ready to directly, deliver this. Now. I would like to discuss the, different forms of guide RNAs, and improvements. We've specifically, made to the chemically, synthesized, versions, that we offer at IDT. Initially. A common, way to introduce, the guide RNA component. Was, by in vitro transcribing, this single, guide RNA from, a DNA template, and then, introducing, this to yourselves. This. Can result in successful editing, however. It is often accompanied, with large-scale cell, death and here. In the, far left pant, panel, I'm. Showing HEC 293, cells that, constitutively. Expressed cast. Nine but. There's been no treatment, administered to these cells these, cells have been allowed to grow to confluency. Over 48, hours the. Middle panel has. 3d nano molar of an in vitro transcribed, single guide RNA transfected. Into these cells while. The right panel has, 13 animal or of a chemically synthesized, and chemically, modified, guide, RNA delivered, now. You can see in the middle panel the, delivery, of the in vitro transcribed, single guide RNA was quite toxic and the cells have undergrown undergone. A lack of cell proliferation. However. The chemically, synthesized, and modified. Guide RNA delivery. Has not affected, the cell growth at all on the right panel. Looking. Further into this we, published a paper earlier, this year showing. That unmodified. Guide RNAs either. In synthetic, form or in in vitro transcribed, form induce. An interferon, response. Now. Here we are comparing four different guide RNA forms delivered. Into human peripheral, blood mononuclear cells. Now. If you draw your attention to the table in the top right corner, I'll go through the four different rnase, that are used in this experiment. Sample. A is a. Guide RNA that was chemically synthesized, but has no chemical.
Modifications. Included, no nucleus, nucleus. Stabilizing. Chemical, modifications, this is an unmodified chemically. Synthesized, RNA. Sample. B is. Also chemically synthesized, but we have introduced, chemical, modifications. Such. As to primal methyl bases and phosphorylate, linkages, to stabilize, this. RNA. No. Sample3 is an, in vitro transcribed. Single. Guide RNA that, still has the v prime triphosphate, intact. And. Sample. D is also, an adventure transcribed, single, guide RNA but, we have we have phosphatase, treated this to remove that v prime triphosphate. Now. To walk through a section. Of data from this work. Please. Draw your attention to the left area. Of the screen where we have the black bars here. We are looking at total, editing, of the four different guide RNAs we, were looking at compared to a positive, control and what you can see here is all. Four, versions have identical, total editing in this cell type whether, we are delivering these as in vitro transcribed, rnas or as chemically, synthesized, guide RNAs with or without chemical, modifications, so in this case we. See, identical. Editing. However. Now if you move to the second figure we. Are directly comparing, here, sample. A which, is a chemically, synthesized, guide RNA with no chemical, modifications, to sample B which, is a chemically synthesized, guide RNA containing, chemical. Modifications, and what you can see is that sample, a the unmodified RNA, has, induced, has, induced, significant. Levels of interfere and alpha while, the RNA. That has chemical. Modifications. Included, in the, guide RNA has not induced, any level, of interfering alpha. Now. Moving to the third panel, we. Are now comparing, sample, a which, is again an unmodified. Chemically. Synthesized, guide RNA now, to an, adventure transcribed. Single guide RNA that contains the 5-prime triphosphate, and now you can see that, the single guide RNA which is also unmodified. Has. Also induced. Interferon. Alpha levels into, a slightly higher degree, than the chemically. Synthesized, form. Now. In the last panel on the right we. Are comparing the in vitro transcribed, guide RNA that contains the, 5-prime triphosphate to one that has had that removed now, you can see removing the 5-prime triphosphate, does reduce the level of interfering, a to, a significant, degree however. This there is still detectable, levels, the. Only time no interferon, response, was detected, was when we use a modified, chemically, synthesized, guide, RNA. Now. As I just discussed a large. Benefit, of using chemically. Synthesized, guide RNAs is the ability to include, chemical. Modifications. That will reduce the risk of triggering the cell's innate immune system. But. Adding chemical modifications. Also. Provides improved, nucleus, stability, to the guide RNA which, results in increased efficiency. Of the editing. Another. Benefit, of using chemically. Synthesized, guide RNAs is that. You will have high quality experiment.
Ready Reagents. Without, needing to do any work in your lab, as. I mentioned the, individual, C RNA and tracer RNA can be synthesized. Separately. And then, annealed in your lab and this, is the most cost-effective, option because. The smaller RNA, the C RNA contains, the target specific, region and can, be synthesized, by the thousands. And can even be used for library screens. This. Then as I've, mentioned needs, to be annealed to the tracer RNA which. Is universal, and this can be ordered in large quantity, amounts and stocked in your freezer if you. Do prefer to have the two components, two, components. Synthesize together as a single guide RNA that is also an option. In our lab we have looked at hundreds, of chemically modified, RNA Allah goes to, determine which bases, of both, the C RNA and the tracer RNA regions, can, be chemically modified, without harming, the activity, of the guide RNA. So. Here, we're looking at on write it on, the right hand side the, C RNA and the. First portion shows, the target specific, region the 20 base protospace or guide domain and then, the, right hand side is the 16, base binding. Domain which is universal, and, what. We are showing in red are, bases. That are allowed, to be modified, with, 2 primal methyl bases we've also looked at other 2, prime modified, bases, without. Any loss in activity, now. If an arrow is indicated, above the base a larger. Arrow indicates, a major loss in function if we placed a modified base at this position where. A minor loss, was shown and was more of a sequence specific effect if there is a small arrow. Now. On the left is the, tracer RNA and we also show here, which bases in red are allowed, to be chemically. Modified, without losing, any activity. Of the guide RNA and. Again with large arrows bases. That need to remain as an a natural, RNA, base. So. We, have taken what we've learned there and, we. Now have three, different options of chemically synthesized, guide RNAs that we offer at IDT.
The. First option, on the left the alt r2 part is what we've been offering, for 3 years now this. Is comprised of a 2 part system where. In green is the, C RNA which, is moderately, modified and, the, trace RNA, which is pretty heavily chemically. Modified, based. On what I just what we just learned in the previous slide that I showed you and all, of the RNAs we offer at IDT all contain, chemical modifications, it's just different degrees, of how, high that modification. Is and. I'd, like to walk through now. Specifically. What these different forms are and when you would want to use each form so. As I mentioned on the left are standards system, which, we've been is what's, been offered for ever's has. Moderate, chemical, modifications, and this works great in systems, where a cast nine is already expressed and also works really great when delivered as an RMP complex, for. Most sites, now. Our new systems, in middle. In the middle is the. Altair XT, two-part, system. Now. This is also a two-part, CRNA. Trace RNA that needs anneal together. But. Now the C RNA has, an increased. Level of chemical modifications. More the bases have these chemical, modifications, these nuclease, nucleate stabilizing, basis but. The tracer RNA is the exact same trace RNA, we had previously been offering so, if you already have this tracer RNA in your lab you. Can just if you want to try out the more modified see RNA this. Is the same trace RNA we've always been using it's just now anneal, to a more modified c RNA and. Then. The far, panel on the right is we. All now offer a, RNA. Molecule, where, the C Renee and Tracy RNA portions are fused together so, a chemically, synthesized, single guide RNA which, is 100 nucleotides, in length requires. No annealing. Has. A moderately. High level. Of chemical modification. So. The two forms on the right work. Well also and, the, advantage of using these, are, if, you, are Co delivering, these with cassadine in an expressed, farm so, when, the rnase need to remain in the cell for longer while, waiting for Cassadine protein, to be made and I'll walk through some experiments, that highlight that in the next slides, these. Also work well with, RMP, and can. Have a slight advantage over the standard two-part, system which only has a moderate, level of chemical modification. If you're working in difficult, experimental, conditions, with, for, example high nucleus, environments, and. I did want to point, out that all three of these options have.
A Three to five day turnaround, time so. You can get these experiment, ready reagents, pretty. Rapidly to use in your lab. So now I want to go into some data where, I compare, the. Three different forms of guide RNAs that IDT, offers and I, want to look at this in the context, of the caste 9 source that you're looking at. So. To do this we, have looked at 12 different guide, RNA sites that target hprt. And in, this slide we are delivering these into HEC 293. Cells, the. Electroporation. We. Are taking the 12 different guide RNAs that, were synthesized, as a standard, two part as the. More modified, 2 part the 2 part XT, or as, a single guide RNA and we have complexed, each, of these 2 Cassadine protein and delivered, as an RMP complex, with. The guide RNA at a one point two to one ratio with the Cassadine protein, and we're also including, the electroporation. Enhancer, which I will go into more detail in the next slide. We're. Also taking all, 12 of the guide RNA sites. Synthesized. In the three different guide RNA forms the, standard two part the, two part XT and the single guide RNA and Co, delivered these with Cassadine plasmid, or cast Knight mRNA, and what. We're looking at is the presence of insertions, and deletions by, NGS. So. Now to look. At the data if you draw your attention to the top left corner. We. Have now delivered, the. 12, different guide, RNAs in, the, first section, as a standard. Two part and this is shown in a violin plot where we've got the sum of all the editing for all of the sites shown, and as the plot. Gets wider, that's where you have more of those levels. Of editing represented, and. The white dot shows, the mean, editing, of all the sites but, what's important to see in the top left corner, is that. All, three guide RNA forms have the white dot at the. Same level of editing so you're not getting any advantage, when you have delivered these with, cast nine RNP. Delivering, these as an RMP complex, when you're using the more modified, guide up guide, RNA options. Now. Moving to, the top right, panel where, we are now delivering this also as, an iron key complex, but we have dropped the, concentration. 12 full so the guide RNA is now at point three micro molar where it was initially at four, point four point eight micro molar so. Going to a sub optimal, level of delivery what, you can see again now is that, the three guide, RNA forms have. Virtually, the same, level, of editing, so you're not getting any increased, editing here by using the more modified, two, part or the single guide RNA. However. Now, if you go to the bottom, left corner where. You are delivering cast nine in an. Expressed, form as either a plasmid. On the bottom left or as an mRNA on the bottom right you do, see a mark a market, improvement, when using the more modified, two part or the single guide RNA and. As I mentioned this, is because the, RNAs are required, to, remain, in the cell for longer while cast nine is being transcribed and, translated so. The more modifications. Render, these more nucleus, protected. And in. These cases you would want to use these more modified. Versions, to achieve higher editing. Now. Here, we are looking at virtually the same experiment. However. In Orange I have, now included. Another. Cell type we, are now looking at, k562. Suspension. Cells and these. Have been introduced, on the Left panels, the top right where this is delivered, as an RMP complex, in the bottom left where this was Co delivered with cast nine plasmid, and what, you can see again, is if the.
Three Different guide RNA forms were, delivered, into. In this case k562. Cells, as an, RMP complex, there, is no increase in editing, by using the more modified, or the single guide RNA fusion, forms but. In the bottom left again which is delivering. These guide RNAs Co. Delivering with cast nine plasmid, we see an advantage of at, using the more modified forms. So. Everything I just showed you was in standard, cell types at 293. And, k562. Cells. Next, we wanted to look at the efficiency, of the different guide RNA forms and cd34. Positive, cells and we did this in collaboration with, aisle the aisle handles, lab at bar-ilan. University. Where. Their goal is to efficiently, knockout genes associated. With, severe combined, immunodeficiency, z' and to identify the best guide RNA form to do so with. Delivery needs as an RMP complex. So. The methods here, involve, electro, / 18 into cd34, a positive, Hamato poetic, stem, and progenitor cells, using. The, all-star cast 9 nucleus, with. A vitro. Transcribed, single guide RNA so unmodified. Or. With, the standard 2 part system the more moderately, modified, or. Our, new highly. Modified 2, part system the, ultra, rxt system and. Also. With a single, guide RNA. All. Of these are MP complexes, are being. Tested with and without our alt, are cast 9 electroporation, enhancer, and what, this is is a, hundred nucleotide, single-stranded. DNA, that. Is non homologous, to human, mouse and rat genomes, but, increases, the electroporation. Efficiency. Of the rmp complexes. One. Other thing they are looking at the end these experiments, is titrating. The amount of RMP, delivered to find the lowest level to achieve the highest editing, in these primary, cells. So. The first target. They're looking at here is reg 2. Now. As i mentioned they are delivering, the RNP complexes, in increasing. Doses so. If you draw your attention all the way to the right where, it says 4 micro, molar, here. We are delivering, the. Guide RNA as either. An in vitro transcribe single guide RNA as the. Standard two-part, as the. More modified 2 part or as a single guide RNA, now. You can see at the 4 micro molar dose the high dose and the.
Dark Red bars there. Is. Little. To no difference, in total editing, just like we saw in the 293, inkay five six two cells as these, are being delivered as an RNP complex, you do not see any difference, in editing levels if you are using an unmodified or, more chemically, modified, versions, and. This is true, walking, down all the way to the left part of the graph when you are delivering these at, 0.5 micro molar at a suboptimal dose, you again still see very similar editing, across, the different guide RNA sites, but. What is also conveyed, in this figure and the, lighter pink bars is the. Presence. And absence of, our electroporation, enhancer. And what, you can see and the light pink bars are when these are MP complexes, were electric rated without the single-stranded, DNA present, or in the dark red bars when. The RMP complexes, included, the electroporation, enhancer, and, you can see how the electroporation. Enhancer, significantly. Boosts editing levels for all of the guide RNA forms in the, cd34, positive, cells. Now. Here, we are looking at a second target rang, one with. The same experimental, conditions. Now. Again if you draw your attention, to the far right of the graph the highest dosed of RMP delivered in this case which was 8 micro molar, you. Can now see that. We. See much higher on target editing when we're using the more modified, guide. RNA forms both. The two-part X T and the, single guide RNA have, increased, production of indels I showed. In the previous slide that. All guide RNA forms had similar on targeting, editing, levels as these are delivered with RMP and so, this set is also delivered with RNP but. It is worth pointing out that we do see a small, subset, of sites especially. When we're working in these, higher nucleus, environments, that. Additional chemical. Modification, can be helpful to achieve, higher editing, even. If delorean with our NP this. Happens to be one of those sites. So. If, you have a site that you are delivering as an unmodified guide, RNA or more moderately, modified, guide, RNA and you're not achieving the level of that you would like it may be helpful to use more one of these more modified, versions. What. This slide also shows again for this target reg1, in dark. Green is the, inclusion of the electroporation, enhancer. While. In light green is the absence of the electroporation, enhancer, and again you, can see including, the electroporation, enhancer, significantly. Boost a total editing for all guide RNA forms. So. The first half of my talk was focused, on looking at on, target. Editing levels of the different guide RNA forms. Now. I want, to switch gears slightly and, look, at off target analysis, it, is becoming, increasingly. Clear the importance of assessing, off target analysis, assessing. The off target effects of your CRISPR cast nine components so. Specifically, here we want to look at if there are any differences, associated, with the, form of guide RNA you use as well as the form of cast nine.
It. Has. Been pretty well published, now that, off target effects are concerned, and that delivering. Cast 9 and in Express form. Does. Increase the risk of off target effects due to the continued, and long lasting of, the, cast 9. And. That delivery, and cast 9 as an RMP, reduces. Off target editing because. The RMP is rapidly, degraded and doesn't remain in the cell as long as the expressed, forms, however. We. Have found that while RNP, does greatly, reduce off target effects there, are some off target sites that do persist, and are, still a problem. So. What do we do about that. There. Have been there's, been a lot of work done trying, to mitigate this and work. Such as reducing. The length of the C RNA to make it more specific. Introducing. Chemically modified bases at certain positions to make things more specific, and while. These works sometimes they also do, reduce the on target editing and then there's also been a lot of work in looking at, making mutant, forms of cassadine that have higher fidelity. There. Have been many great publications. Discussing. Various. High fidelity mutants, that, were developed through rational, design, but. These, were evolved, under conditions, where caste 9 and the guide RNA were, expressed, as plasmid. And we, have found when testing these forums, that delivering these an RNP form does, reduce off target effects but we also see. A significant. Reduction in, the on target, activity. So. We. Sought out to, develop a protein, that would maintain on, target activity while, reducing off target activity specifically. When we deliver these as an RNP complex, and, I'm not going to go into a great deal of detail here but. We did this by, using a dual screening, approach where. We selected, for maintenance of an on target, activity and, absence, of off target activity where. We have a high copy plasmid, on the left that. Expresses, a bacterial, toxin, on the, on target site so, this needs to be cleaved to survive and on, the Left we have a second, plasma, plasmid, that, has an off target site that, expresses, an antibiotic, marker and we, need the plasmid, to not cleave so, we. Made a random mutagenesis library. Of cast 9 and pass it through the screen and assess, our positive hits. And. The. Mutant, we ended up settling on is discussed. In our nature medicine manuscript, that was published last month where. We show that we've we've. Identified a, high fidelity cast, 9 mutant, that, when, delivered as an RMP complex. Maintains. On target activity but. Also reduces. Off target activity and we show this in standard cell types as well, as in human Hamato poetic stem cells and. This. Is referred to commercially. As the Alta our high fight cast 9 and is available through. IDT and. If. You want any more detail about that protein this is the publication, that highlights all of that I. Did. Want to show one, key figure from this paper, just. To show how this, version does, maintain, on target, activity, when. Delivered as an RMP complex. So. What we are looking at here are 12. Different guide RNA sites and we, were looking at just total on target activity here, and we are comparing, this to on, target activity of a wild type cast 9 again. Delivered as an RMP complex, so you see those levels in blue and, then. If you go to the end of each.
Set. In green, is the alt our high 5 cast 9 and so, what you can see as you walk across the graph is that, the, new. IDT. High, 5 cast 9 has. Very, similar on target editing levels when delivered as an RMP as a wild type cast 9 well. If you look at the other. Forms, the other published versions that were evolved, for cast 9 plasmid, there's, a market, hit and on target activity in the orange gray and yellow bars from, the majority of the site's. So. We. Know off target, effects, can be an issue and we've evolved, a new protein, to help mitigate this but. We wanted to now look at if the different guide RNA forms have, any difference, on the level of off target effects, so. The next set of data I'm going to show you is looking. At the, most common, forms, of guide, RNAs and assessing. If these have any difference, on the levels of off target effects that we detect so. If you look at this table in. The top line we, are we, will be looking at a plasmid. Single guide RNA so delivering the guide RNA in an expressed, form and, the. Next three options are chemically. Synthesized, two-part guide RNAs either. As completely unmodified. As I've. Talked about in. Great detail our standard, two part which is moderately, modified and, then, the fourth line down, the. Two part XT, which, has increased levels of chemical, modification, now. The last three lines in this table our. Guide. RNAs, that are synthesized. As a single, guide RNA as one RNA molecule. So the first of those three lions and in vitro transcribe single guide RNA which, is made through enzymatic synthesis, the. Still has the triphosphate intact, and, then. Comparing this to chemically. Synthesized, single guide RNA set or unmodified, or, the IDT, chemically. Modified, version. Now. Before we show the data I do want to take a step back and discuss how to identify potential. Off target effects which sites should you be looking, for. There. Are methods to predict or validate, after there are many methods out there to predict or validate off target sites and there are a lot of in silico prediction, tools also a lot. Of these currently. A lot of these in silico tools do. Actually miss a lot of important, sites and can also over predict sites so it makes it a challenge to then if you're going to do amplicon, sequencing, and assess, all of these off target sites to know which ones to look at and to have a manageable, level, to. Actually study, there. Are also. Been publications, of different in vitro assays, to define off target effects but this is not unique to your cell type and can also often be over predictive. The. Guide seek approach is. Commonly. Used and. This. Is where. You empirically, determine, off target sites in your cell type through an unbiased approach, but. This is more of an identifier, and not, all really quantitative. In, a. IDT, we. Have developed the. Ramp seek system for CRISPR that, we use in-house but, this is not yet commercially available and, what we use this for is after, identifying, what. Potential, off target sites you have via, guide seek we now go and do a multiplexed. Amplification. Based target, enrichment, approach where, we can look at all of the off target sites that we've identified through. Either in silico prediction, tools or unbiased methods, and actually. Quantitate the level of off target effects were seen in our, delivery systems, so we are able to, include. An on target site and up to 1,000 off target sites in a single multiplex, reaction, and this is known as ramp seek for, CRISPR which we currently use internally. And this, will be commercially available later, this year. So. The two sites we were going to be studying in the comparison, of the off-target effects for the guide RNAs are AR, and AMX one, so. First we need to determine the potential off target sites that we want to look at for these two targets so, I just kind of wanted to highlight the workflow, that we follow in our lab so, the, first thing we do is we, do we use the guide seek approach where, we deliver our guide, RNA into. Cells that Express caste 9 along, with the guide seek double-stranded tag and identify. Which, where. The where the double stranded tag is being integrated to us to. Identify potential. Off target sites and. As I mentioned we are delivering these into cells that Express caste 9 and we, do this because as I have mentioned. Delivering. Caste 9 in an expressed, forum has higher off target effects so in this case we actually want to go into cells that have caste 9 expressed. To get a be, able to cast a wider net and assess, any. Potential, risk that we have for sites, that could be, mischief and cosmas, Shiv and have an off target effect happening. Because. Of this we also use our more modified, to part system again, because now this RNA is going to be more stable, so this basically just gives us the highest chance of finding all potential, off target sites, so.
The Panel on the right shows, a typical, readout, we will get after doing guide seek through. The, NGS, analysis, and a. Does. Sites are all identified that have detectable. Reads of the, guide seek tag after making, our double-stranded, break we. Also then do add on into this list some, in silico prediction tools that have three to four mismatches, and we, take this list and, we does. We have. We, design and synthesize, a ramp, seek panel where, we can then multiplex. All of these sites together in one library prep so. For the two sites we'll be looking at today, AR, has. 54, assays included, in it the on target assay and 53, off target sites that we've identified and, this means an. E/m x1 which, has 32, assays so the on target assay and 31. Off target assays that we've assessed through this workflow. Now. Here, I do want to show why, we are deciding, to use our more modified, two-part, system when, we do the guide seek identification. Approach. And what. You see here in the, top panel in blue. Is when. We have delivered, the. Two-part. Xt guide, RNA into cells that Express caste 9 and we, are assessing, off target sites and via guide seek and every bar here represents somewhere. Where we have detected, an off target site so. The top panel is looking at three different guide RNAs in blue. Where, we have delivered, a more modified, two part and below this is the same guide RNAs, but, in green is where we have to deliver, it a less. Modified, chemically. Synthesized, guide RNA and what, you can see here, is. That we are identifying more sites when we're using a more modified, guide RNA again, because this, guide RNA is going to be more stable and. The, black numbers, the, percentages. Represent. The sum of the total unique, reads that we're seen so, what this shows is, that, the majority. Of the reads that we are finding for the two different guide RNA forums had a high level of overlap, the, more modified XT, form, just. As identifying, more sites so again this gives us the greatest assurance that, we are now going to. Use. Amplicon. Sequencing, and assess, the highest risks all potential, risk sites for off target effects. So. Now to move, into the experimental, details of, looking at the off target effects of the different guide RNA forms we. Have transfected. All of these different guide RNA barians that I discussed. Targeting. AR and AMX one into. Cells. That Express caste nine and, then. We have also delivered these as our MP complexes, where we've taken the different guide RNAs and, complex. These two caste nine nuclease in either wild, type or the, all tar high form so now we are able to look at the, difference enough target effects of the different guide RNAs when, delivered into cells that Express caste 9 or, when delivered, as an arm P Complex in either wild type or high-fidelity, form and anytime, we are introducing an RMP, complex, we are including. The alter electroporation, enhancer, and, then. We, are assessing, now on an. Off target editing levels through the ramp seek system where we have panels defined based, on the previous work using guide seek to identify the off target sites for these two targets, AR and AMX one. Now. There's a lot of data in this slide so I'm going to try to walk you through this in a, pretty, detailed form, so. This. First target, shows all, of those experimental, conditions, targeting. AR, so. If you look Horace. On Thalia cross these graphs and the, things, that are boxed off with each of an individual, pie chart these, are the seven different forms, of guide RNA and. As. A plasmid, single guide RNA as an unmodified and, etc and now, these seven. Different guide RNAs are, delivered. Vertically. Into, cells that Express cast 9 in panel a or. Delivered. As an RMP complex, in panel B as a wild type cast 9 boring. Panels see with, the high fidelity casting, now. If you draw your attention, to panel. A I'd, like. To walk you through what all of these bars mean, so. The first bar in orange. Is the on target, si so as I mentioned these panels are designed to have the on target si and all of the off target assays included, in them and. In blue I'm showing the top 10 off target sites we did run the full panel but this is just showing the top 10 sites here and then. The. Pie chart shows. You the focus, here is to look at the orange region and the number there because that's the total reads that, are on target, so you can see for example. The. The. Single guide RNA at the far end of panel a the. Majority of those reads actually, are, off target effect reads so there's a high level of off target if any are, off target editing associated. With this guide, RNA site, so. If you look across panel. A and look, at the numbers above, the orange bars. What we're seeing here is that we're seeing similar on target levels, of editing when we're delivering delivering, all of these different guide RNA forms, and.
Then If you look at the blue bars you, can see that all of these guide RNAs have when, delivered into cast nine cells do, have pretty significant. Levels of off-target effects and. If. You, look at the alt rxt. In the. Middle which. Has 34, percent of the reads as on target so the majority of the reads are off target or. If you look at the single. Guide RNA at the end of the panel a these. Two forums which are the most modified, do have slightly higher off target effect levels and this makes sense because these are going to remain stable and the self route longer, but, across the board delivering. All, of these different guide RNA forms into Cassadine cells there, are pretty significant, levels of off target effects, now. If we move down to panel B and we, look at the orange bars we. Can see that there are similarly, similar. Levels of on target editing. The. Unmodified. Has slightly less and, in this case a single guide RNA has slightly higher however. We've, made a market, reduction, now in delivering, these. Guide RNAs as an RMP complex. So. As I mentioned before switching. To RNP is going to greatly reduce your off target effects but you can see there are still some blue bars that creep up but, now the majority of the, reads, that we're detecting are ninety. Percent and greater on target, any. Off target sites are, below one percent of the total reads but. They're still present so now if you move to panel, see what's. Worth pointing out first is that the numbers above, the orange bars are virtually identical to. Panel B so as I mentioned we developed, this protein to maintain. High, on target activity and this nicely shows how that is the case we were seeing very similar on target activities, when we were delivering the, high, fidelity cast, nine as when we are delivering the wild-type rmp complex, but now we have completely, removed all off target effects associated. With any guide RNA by, using the ultra, high fidelity new nuclease. Now. The second, target this, is set up in the exact same way. Is looking at a mx1 now this is a, popularly. Published. Target, site because it does actually have pretty, significant. Levels of off targets, and so, now if you look at panel a again. Where. Cast nine we. Are delivering the different guide our guide, RNAs into cells that Express cast nine you, can see again. Very high levels of off target effects associated with all guide RNA forms and in this case there's not really a certain, guide RNA that stands out as having more off target effects there they all have pretty high levels when, you're using cast nine in an expressed form, so. Now. Moving down to panel B again. Take note of the numbers above the orange bars where, we're seeing similar levels, of editing for all the guide RNA sites, so we're not biased there, but. Now. The. Blue bars again, now actually, switching to RMP there, are still pretty significant, levels of off-target effects. 60. To 70 percent of, your, reads are on target but there's a good portion that are actually, off targets, so this is what I mentioned that there are some sites that do persist even if you switch to RMP delivery. So. Now switching. To high fidelity cast 9 again.
The Level, of editing of the numbers, above the orange bars are virtually, identical to the wild type delivery, but now we have virtually. Reduced almost. All of the off target edits, associated, with this very promiscuous. Guide. RNA site. And. It's. Also worth pointing out, that all, of the seven different guide RNA forms, that. We delivered as well, as three different casts nine forms, produce. Identical, repair, profiles, so. If you just, focus your attention on panel, B what. We are looking at here is the, individual, bars are the seven different guide RNA forms for, this particular site, AMX one and. The zero mark is anything that we sequence, that was left at wild-type so we saw you know about 85% editing, for the majority of these sites but. Then what we were seeing is that this, site has a repair, profile, that has a very predominant one, base insertion. And then, a, negative. Minus. Three delete, three bass deletion, or a six bass deletion, and what, is really interesting, to see is that all of the different guide RNA forms have, the identical repeat profile, and panel B and now if you look at all. The, panels as a whole you. Can see not, only do the is the, repair profile, the same for the different guide, RNA forms it's also the same for the different casts nine forms so now delivering, these, into either cast nine cells or switching in using our high fidelity mutant. You have that we have not changed anything with the repair profile. So. To wrap up the data that I've showed you there. Are many different guide RNA formats available and most, of these gives similar on target editing levels when, they are deliberate as RMP, the. Higher modifications. Though do. Have. A nice advantage when you were Co delivering, the guide RNA with cast 9 and X in an in an expressed, form and. There. Is a subset, of sequences, though that, do respond, better to higher, modification. Even. If you're delivering this these as R and P and this, does tend to be a sequence, and cell type dependent, effect I, went. Into great detail about the benefits, of chemically synthesized, guide RNAs having, reagent, having. Experiment, ready reagents, but also the ability to add in chemical, modifications. Which, increase stability and, reduce, the risk of triggering the cells innate immune response where, unmodified, guide RNAs are in vitro transcribed, single guide RNAs induce those high levels of, things. Such as interferon, alpha. Then. Moving to the off-target, effect analysis, I showed, that the different guide RNA forms really. Don't result really, result, in similar off-target editing, levels, there's, a slight increase with more modified, versions, but really it's the source, of caste 9 that drives the. Oft targeting, at the off target editing, so. Delivering the guide RNAs as an RNP complex. Does, show a huge reduction in nonspecific. Editing but, getting using. The ultra. High fidelity cos 9 really, further reduces, any of that off. Target editing. So. I just wanted to take a minute here at the end and, showcase. All. Those all of the different tools we have, now developed in. The research group at IDT. Four, nice, CRISPR tools, that. Is now a complete, workflow, essentially. Where, if you look at the Left panel. We. Now offer a CRISPR, cast 9 design, tool that, has pre-designed, guides custom. Designs and, there's also where, you can check your existing, designs and this works for human, mouse rat, zebrafish, and C elegans and we, will soon also be adding an HDR, design tool to this. To. Make your cut so. You need, your guide RNA and your proteins, for, the guide RNA which, we've talked about in great detail today. We. Have the, two-part system as either the standard two part or the more modified. We. Have the chemically, synthesized, single guide RNA and then, we also have the C RNA for the cast weld a and as I mentioned all of our guide RNAs all include chemical, modifications, just the, XT, has is more modified, than the standard. We. Had a webinar earlier this year talking.
About The different. Proteins. CRISPR proteins we have and the improvements, we've made to increase. The efficiency, of these, are. Current suite includes, the wild-type cast nine high, fidelity cast, nine both. NICUs versions, and cast 12a and earlier, this year we went from our v1 version to v3 which. The. Activity, of these has all now increased, and. As I talked about today we have. The electroporation, enhancer, which includes, which, increases, the efficiency when, you're delivering your. RMP, complex. The. Electroporation. And we. Have electroporation, enhancers specific, forecasts nine and cast well babe now. The majority are all of everything I talked about today was looking. At the non-homologous end joining repair, what we were just specifically, looking at in Dells we. Do have a suite. Of options for, if you are wanting to add in a donor, template, to make a correction we. Have Alzheimer's. Which are single stranded DNA s that go up to 200 basis, and we also have mega mer single-stranded. DNA fragments. Which can go up to 2,000 bases and, what I'm going to talk about lastly, on the next slide is our newest reagent, the alt our HDR, enhancer, and then. Lastly - now, you have, designed, your experiment, you have major double-stranded, break you have, maybe introduced, a donor. Template, to make a repair now, to analyze. Your. Editing, we. Offer the, genome, editing detection. Kit which is a t7, based system which is works great for simple screening, of your. Components. But, as I mentioned today we also have developed. In-house the, ramp seek system for CRISPR which is a multiplex. Amplification. Based. System for Illumina 6 sequencing, that allows you to look at up to a thousand assays at once and, this will be available, commercially, later this year. So. I did just want to end on talking about our newest - reagent because this is really exciting we, launched this last week, we. Launched, a H, we now have available an HDR enhancer, which. Is a small molecule compound. That increases, HDR, efficiency. From. Here I'll just show one slide of data or, I'm showing, that we were able to increase HDR, rates with, past, 9 and past 12 a nucleases, by, including the HDR enhancer, in the experiment, so. But, left to the left of the line the, first 4 sites what we are looking at is a, HDR. Rates for four different sites when, delivered. Wendel. Using, the cast nine nuclease, so in blue we, are just looking at HDR rates of including, the, RNP complex, and a donor template, but in orange we have now so now, also included. The, HDR enhancer, and this is done in jurkat cells here but we've seen this in many cell types and. What you can see is that the orange bars actually increase, the HDR rates in some cases by almost six-fold. Now. The same is true on the left where we are now looking at for sites that have, a. Cast. 12 8 p-m site so we are now delivering our MP complexes. Forecast. 12a and including. The. HDR enhancer, and orange or just looking at the the, natural, DR rates and again you can see here the market improvement, of including this HDR, enhancer, so, this is now available on our website and is an exciting new product that we have. So. To wrap, everything up I just. Wanted to put up some a couple take-home messages about the different guide RNA forms because like. We've seen today there are a lot of different options. So. The standard, two part system like I've said works very well for many applications and. Is the most cost effective synthetic. Option. This. Works great if you are working with cells that Express caste 9 it, works great as a tool for screening. Guide RNAs and even for looking at gene libraries, it works. Great with RMP, delivery, the, majority of the time and they've actually been reports, that the two part system can, have greater efficiency. Than single guide RNAs in some systems such as zebrafish, and. Then. As I've discussed today we have a two part system that has increased, chemical, modifications.
The Alts are xt 2 part and we. Also have a single guide RNA and so these two options which have an additional, nucleus, stability. Have an advantage. If you are Co delivering, these with a caste 9 plasmid. Or mRNA if you. Are delivering these with lipid nanoparticle. Delivery, if. You are working in high nucleates environments, and like, I said there is a subset, of sequences, that are more susceptible to nucleus, degradation so. Adding, more chemical modifications, can sometimes really improve your editing. So. That wraps, up my portion, of the talk today and if you want any more information, we. Have a lot of really good information on, our website, we have a lot of posters and, other webinars, and we have a lot of internal. Generated, protocols, as well as protocols, provided, by some of our users all, available, at the website on the screen. So. With that I want to thank everyone for their attention and I pass. It back over to Sean so thank you. Thank. You Ashley for, that informative, presentation. If. You have a question, and have not done so already please type, it into the Q&A box. We'll take about five to ten minutes for questions now so, here's the first one. Okay. So we had a few questions about our alte our electroporation. Enhancer, that you mentioned, ashley customers. Wanted to know how. The electroporation. Enhancer, works what the mechanism, of action is and then, also if we see. Any stimulation, of the immune system when using the electroporation, enhancer. Okay. Thanks Molly so. The electroporation, enhancer, is included. It is not formed, with the RMP complex, so we formed our inca pea complex and then, we add that to ourselves and, then we add the electroporation, enhancer, and that is all electroporated. Together, and. What, we see is that this overall. Increases, the efficiency of, getting this into the cell now the exact mechanism, we, are still trying to understand, but we, see this as acting as a carrier, DNA, where we were increasing. Shuttling, of getting these components, into the cell and the, electroporation, enhancer, is a nice way to do this another. Option is you can increase, the amount of guide RNA, in. Your RMP complex, where, you're now adding an additional. Nucleic. Acid, in there but, there, really is a nice. Additional. Boost. In editing when you include this and, then. The second part was. What. Are the immune responses. If any of the electroporation, enhancer. Yes. We. Have not looked. At that in as great of detail as the RNA but the. Work, we have done show, very, little and you neurons of this we. Have done, a lot of work to optimize. The sequences, that we are using for. These DNA. Sequences, of what as well and we've also looked into the rates, that this would integrate into your genome and those rates are also extremely, low, great. Thank you. We had another few. Questions, about, if. You're able to the RNP complexes, and other systems, such, as fungi, or plants. If. You have any recommendations. On that yes. There, have been some publications, now. Specifically. With plants, that the RMP system. Is very efficient, with so. We have definitely, heard positive feedback, there I know there was work done trying to understand if this will work in bacteria, and I haven't seen a. Lot. Of publications, on that yet but I know that's an area people are actively working on great. And, one researcher, wanted to know if the guide seek results, that we showed in the HEC 293, cast nine staple cell line do. You think those would be representative, of results, in other primary, cells that we're expressing casa 9, yes. And we have actually compared. With. Some collaborators. Cast. Nine cells and different cell types and we do see very similar levels, of which. Off target sites are seen across. Different, cell types, great.
Thank You. Okay. Here we have a question about that HDR, enhancer, that you just mentioned a researcher. Is wanting to know how it compares, to other small, molecules tested, such as SCR 7, so. We actually we. Did a before, launching this we did a very thorough screen, of many. Different small molecules, and this is actually the only one that we saw that gave this market, improvement. We. Saw that it had a very. Much. Higher increase, in editing over any of the other published. Molecules. Out there great. Do. You have any data on human, embryonic stem cell, editing, for. Example what is the best combo, of reagents, that could be used for these cells. We. Actually on our website do have some protocols. For, that, particular cell type that I would direct you to look at specific, conditions, there but we, have seen, very high levels, of editing with using. The modified, chemically guide rnase complex. To the Cassadine, protein, both the wild-type and the high fidelity. Great. Thank you, these. Are great questions thank, you for writing. In everyone, here's. Another one so do you observe any effects of, the RNA, secondary structure, on the. The efficiency, of editing. That's. A good question. We. Considering. The trees. RNA and the RNA, does have a lot of secondary, structure, we. Have not noticed any decrease. In activity with, using the single guide RNA this is as efficient. As the two part system. Great. Okay, and I think we have time for just one last question we'll, try to get back to any. Other questions, in. The future, but. The final, question would be do. You have modified, guides, for the caste 12a, system, as well as the caste nine system, yes. That's a great question and as. I mentioned very briefly at, the beginning, this. Focus of this presentation was. Cast. 9 we do have webinars. Available on our website that specifically. Discussed, caste Apollo Bay but, we, do have, the, caste 12 a CRNA, so for Castro Bay it's just a single, short guide RNA so the C RNA is about 40 nucleotides. In length and we have studied. Again. Hundreds, of different sequences with various chemical, modifications, and what we offer on, our website is chemically, modified, a chemically. Modified caster called a CRNA. Thank. You actually, okay. That is all the time we have for questions I, want. To thank all of you for attending today's presentation. I also would like to thank. Actually. For an informative presentation. As well as Molly for conducting, the question-and-answer session this. Is one of a series of webinars we'll be presenting on CRISPR as, well as other topics we will e-mail you about these future webinars as they are scheduled, also. As a reminder recording, of this webinar will, be posted shortly on our website and at, youtube-dot-com. Forward-slash I, DT DNA. Bio there. You will find several, other educational. Webinars on such topics as next-generation sequencing. Genotyping. QPCR, and general molecular, biology, thank. You again for attending and we wish, you the best of success in, your research.