Agilent Technologies, Inc. (A) Earnings Call Transcript & Summary

Hello, everyone. Welcome to BioProcess International Spring Digital Week, brought to you by the producers of the face-to-face Bioprocess International events, visiting Boston in September and Milan in October this year, and our Bioprocess International Europe will be delivered as a 100% virtual conference and exhibition on July 13 to 17 this year. My name is Eden Turner. I'll be your host for today's session titled Chemically Modified Guide RNAs to Improve Stability and Accuracy. First, I'm just going to cover some quick housekeeping items before we get started. So if you experience difficulties with audio or advancing your slides, please refresh your screen with F5. If you are experiencing other issues, hit the Question Mark button to receive assistance. At any time during the presentation, you can submit your questions into the Q&A window on the left-hand side of your screen. In 24 hours, you'll receive a link to watch the recording of this session. You can also download a few featured white papers in the Resource List box on the right side of your screen. And I'm now going to begin by introducing our speaker for today. We have Dan Fisher, he is the Market Development Manager for CRISPR genome engineering at Agilent. Thank you for joining us today, Dan. I'm now going to hand over to you to tell us more about your guide RNAs.

Thank you, Eden. Hello, everyone, and welcome to BPI 2020. My name is Dan Fisher, and I'm one of the Market Development Managers at Agilent, covering our Genome Engineering portfolio. Today, I would like to talk to you about a couple of product areas related to CRISPR gene editing. One, our chemically synthesized single guide RNA; and two, our CRISPR libraries for screening. Both of these technologies have biomanufacturing applications that we'll talk about today. The title of my talk is Chemically Modified Guide RNAs to Improve Stability and Accuracy. To give a little bit of background on Agilent, Agilent spun out of Hewlett Packard in 1999, at that time the largest IPO with the motto, Innovating the HP Way. Today, we continue that history with their inkjet printer technology, which we'll talk about and also innovative instruments like the bioanalyzer. We continue to grow and develop new technologies in genomics and molecular biology. And we've also acquired a few companies along the way. For example, Stratagene is a strong molecular biology company that has been part of Agilent for over 10 years, and the current team contributed to our CRISPR library. To dive a little deeper into Agilent's current genomics portfolio, it is vast, including NGS, NGS automation, QC instrumentation, CRISPR, Microarrays, PCR and many others. The Bravo NGS automated liquid handling system has been in the news recently with COVID-19 testing, helping to automate PCR. The Bioanalyzer 2100 recently had its 20-year anniversary, showing that it's a real workhorse in the laboratory. Today's focus will be on CRISPR gene editing. Agilent was motivated to move into the CRISPR space because of a breakthrough chemistry, which was developed a couple of years before CRISPR/Cas genome engineering came on board. The chemistry allows for the synthesis of ultra-long, ultra-pure RNA, which is perfect for CRISPR since it requires a guide RNA for locating the target site. Since 2013, CRISPR has revolutionalized genome editing in all fields, including bioproduction due to its simplicity and high efficiency. CRISPR/Cas gene on engineering allows for a variety of applications, including gene knockout, targeted integration, gene activation, gene interference and more recently, base editing and prime editing. CRISPR/Cas genome engineering has transformed the life sciences due to its simplicity and robustness. It's a simple 2-component system that relies on Watson-Crick base pairing to drive specificity. The guide RNA base pairs with the genomic DNA to determine the target site. One can retarget the system by simply changing out the guide RNA. The toolbox is quite vast, including knockout, knock in, interference, activation, base editing, prime editing. The gene-edited cells can be analyzed using a variety of instrumentation, including seahorse in the upper right for metabolomics, LC/MS for protein analysis and sequencing technologies for RNA function. So how does CRISPR work? CRISPR for gene editing is a simple 2-component system. A guide RNA shown in red and an enzyme, such as Cas9, shown in gray. A guide RNA for Cas9 is typically about 100 nucleotides long. The first 20 nucleotides drive the specificity on the 5' end and were the target within the genome. The remaining 80-nucleotide scaffold is necessary for binding the Cas9 protein. You simply mix the single guide RNA with the Cas9 to form a ribonucleoprotein complex, or RNP. The RNP is delivered into the cell, and it finds its way into the nucleus for DNA cleavage. The first 20 nucleotides of the guide scan the genome for complementarity. Once it finds its correct site via Watson-Crick base pairing, a double strand break is introduced in the DNA and the cell can use 2 primary repair pathways to heal the wound. The first is nonhomologous end-joining, shown on the left. The second is homology-directed repair shown on the right. NHEJ on the left can cause insertions due to polymerases in the cell or deletions due to exonucleases in the cell and simply get glued back together, and there is no air checking with NHEJ. NHEJ is inherently a mitogenic process. Therefore, if the guide targets the open reading frame of a gene, it can lead to a gene knockout due to a frame shift mutation many times leading to a premature stop codon. For HDR, on the far right, you can take the same guide and Cas9 plus a third component, a donor template, such as a single-stranded oligo or a double-stranded plasma donor. In this case, you get cleavage by CRISPR-Cas9 and the cell uses the sister chromatid for repair. In HDR, we're essentially tricking the cell into thinking that the supply donor is the sister chromatid, and you get directional synthesis copying in the new DNA sandwiched in between the homologous donor arms. The key with CRISPR-Cas9 gene editing is the double strand break, and breaks by CRISPR-Cas9 induce DNA repair and increase mutogenesis frequencies by 10^3 to 10^5. So the double strand break is key to gene knockout and gene knock in. Bioproduction applications include metabolic selection cell lines such as GS, DHFR, glycosylation knockout such as FUT8 and also knocking out contaminating host cell proteins. Bioproduction applications also include targeted integration on the far right. Specifically, targeted integration is desired in CHO cells for stable protein expression. The challenge has been figuring out which site in the genome leads to robust protein expression. Targeted integration remains a challenge in CHO cells, but it's definitely a desirable trait. There are a variety of ways to introduce CRISPR/Cas into cells. A common way for CHO cell editing is through RNP on the far right, where you have a guide RNA, a purified Cas enzyme, the most common being Cas9. The CRISPR/Cas is electroporated into the cell and the editing occurs in the nucleus, and the RNA and the protein are eventually degraded. For CRISPR library screens, the most common delivery is lentivirus. Therefore, the guide must be synthesized, cloned and cloned into a lenti plasmid. Then the virus will be expressed and then transduced in the appropriate cell line at a goal of 1 guide per cell. With the CRISPR library screen, the guide is integrated into the DNA via lentivirus, and the cell line typically has Cas9 expressed. The lentiviral DNA is transcribed into guide RNA, and then it complexes with Cas9 within the cell. The goal with the CRISPR screens is to deliver 1 guide per cell, so that you only get 1 editing event per cell. To conclude, CRISPR/Cas can be delivered as either RNA, shown on the far right, with synthetic RNA. It's common for targeted applications such as host cell protein knockout. And then the other one is with DNA, with -- being able to go from the printing DNA all the way through lentiviral particles. And this is common for pooled CRISPR screens looking at hundreds or thousands of editing events in 1 assay. Agilent has been a leader in the field of nucleic acid synthesis for over 20 years with market-leading microarrays and NGS products. Our CRISPR libraries are synthesized by our inkjet DNA printing. As I mentioned, Agilent spun out of Hewlett-Packard. Many know HP for their inkjet printers. Agilent has adapted this technology to DNA synthesis via phosphoramidite chemistry for high complexity parallel synthesis of single-stranded DNA oligos. CRISPR libraries can contain thousands to hundreds of thousands of different guides, therefore, making them by inkjet printing is advantageous. If we look on the far right, it shows our GMP-grade oligo. Agilent has expertise in manufacturing DNA and RNA under GMP conditions. We have 2 sites, one in Boulder, Colorado and one in Frederick, Colorado, to produce GMP-grade oligonucleotides for pharmaceutical and biotech partners. More recently, we've moved into a synthetic guide RNA, shown in the middle, due to the development of a breakthrough chemistry with high coupling efficiency, which leads to an ultra-pure final product. The chemical synthesis process starts with a glass surface and nucleotides are added one by one. It's a lot like adding beads to a string, but here, we're adding nucleotides until we get the full-length RNA oligo. Let's look at what we call our TC-RNA synthesis chemistry closer. When we look at the synthesis of DNA oligos, it is very straightforward process to generate a linear biologically active DNA oligo. This is due to the fact that there's only one reactive hydroxyl group that is available for extension. RNA, on the other hand, is much more challenging because there are multiple reactive hydroxyl groups making generation of a linear RNA oligo using native RNA bases impossible. In JACS in 2010, Agilent published a paper outlining a new chemistry that allowed for the generation of long RNA oligos. Referred to as TC-RNA synthesis, this chemistry allows for the streamline preparation of phosphoramidite monomers with a unique 2'-phenylcarbamate protecting group, hence the term TC. This allows for the generation of long linear RNA oligos. However, these oligos still contain the protective group. If you treat these oligos with ethylenediamine, this converts all the protecting group spec to hydroxyl groups, resulting in a biologically active RNA molecule. The breakthrough chemistry -- this breakthrough chemistry yields high-quality biologically active RNA. This is due to the fact -- this is due to the high efficiency per cycle, the high coupling efficiency per cycle. Our routine synthesis is up to 140 nucleotide. In fact, today, we've been going up to about 164 nucleotides, and we think that we can go further. And finally, with chemical synthesis, you can precisely incorporate chemical modifications, which can improve the performance of the guide in CRISPR/Cas gene editing. Here is an example of a synthetic guide RNA at Agilent. It's 100 nucleotides long. So we did the chemical synthesis cycle, and then we run the crude HPLC product -- or the crude RNA synthesis product over HPLC and collects fraction. We assay those fractions by LC/MS to confirm the full length product, and then we pull the appropriate fractions, ultrafiltrate, lyophilized and shipped. The resulting product is ultrapure RNA with purity specs greater than 80%, and we typically see greater than 90% purity with guides that are about 100 nucleotides long. Examples in biomanufacturing include metabolic selection cell lines such as GS, DHFR and also knocking out contaminating host cell proteins. Since our guides are custom, they can be paired with your favorite enzyme of choice, whether Cas9 or Cas12a or whatever your Cas9 flavor of choices. This slide highlights the power of TC-RNA synthesis chemistry, unprecedented length with 162 nucleotide single piece RNA. This system is a little different than the CRISPR/Cas genome editing via double-strand break for knockout and knock in. This is an example of a CRISPR activator. CRISPR activators turn on genes. In this system, several transcription factors work together to turn on a gene. This system works by designing the guide towards the 5' part of the gene. Typically, 0 to 200 base pairs upstream of the transcriptional start site. The guide is fused to a catalytically inactive Cas9, called dead Cas9. The guide is engineered to contain 2 MS2 aptamers to allow MS2 proteins to bind. This complex does not add the genomic DNA, it just sits there with the transcription factors such as VP64, MS2, p65, HSF1 turning on the gene. Since there are several transcription factors working together, the system is called SAM, or Synergistic Activation Mediators. Sometimes it's referred to as the Cas9 activators with SAM. We've heard some impressive data using the system. In an in vivo model, the system leads to 60 days increase in gene activation. On the right, it shows the HPLC trace with the full-length oligo shown at the main peak. After HPLC, LC/MS shows the full length 160 tumor. Looking at the literature recently, Nate Lewis and his colleagues used a CRISPR activator to increase transcription of a couple of genes. They drove the expression of a previously silent glycosylation gene to improve the glycan structure, to make the glycan more human-like. Therefore, there are CRISPR activators used to make targeted alterations in CHO cells for biomanufacturing. In addition to ultra-long, ultra-pure RNA, chemical synthesis also allows for the precise placement of modified nucleotides to improve stability or even specificity. Chemically synthesized RNAs have distinct advantages compared to in vitro transcribed guides. Some IVT guides require the first nucleotide to be a G, and this is not the case with chemical synthesis. It's also harder to scale with consistent purity compared to chemical synthesis. And also, the chemical synthesis allows for the precise placement of chemically modified nucleotides to enhance performance. And here's the list of factors that chemical modifications can affect: Stability and activity. If the RNA is more stable, it will hang around longer, and thus, you'll have higher editing efficiency. The third bullet shows specificity, using chemical modifications to affect DNA, RNA Watson-Crick base pairing such that if you slightly destabilize the hybridization at certain positions in the CRISPR RNA, you can reduce off-target cleavage and only get the desired cutting. The fourth bullet is cross-linking. And then the fifth is labeling, such as with fluorescent dye. On the far right, we see a few -- we see various applications for long RNAs, and we've touched on a few of these, such as Cas9 or Cas9 variants, SAM and also a newer technique called prime editing. Prime editing is a new technique that allows for the gene correction independent of a double-strand break. Essentially, the guide acts to drive the specificity as well as the RNA is used as the donor template for our DNA repair. The system finds the target site, nicks the DNA and then it uses the 3' end of the guide as a template for repair, and reverse transcriptase copies the RNA template into DNA. Therefore, the programmed edit in the guide RNA gets incorporated into the DNA. This is a new and exciting flavor of CRISPR/Cas genome editing. Agilent pioneered the stability enhancing modifications with the collaboration with Matt Porteus from Stanford. These 3 modifications were chosen because of their previously reported stability to serum and snake phosphodiesterases and immunostimulatory properties. In total, 6 modifications were used, 3 in the 5' end and 3 in the 3' end shown in orange. These modifications slow down exonuclease from chewing up the RNA, such that the RNA is more stable, it's going to hang around the cell longer and you're going to get better editing. Specifically, these structures are, we list there as an M, a 2'-O-methyl. MS is a 2'-O-methyl-3'-phosphonoacetate. And then the MSP modification is a 2'-O-methyl-3'thioPACE group. The data shows that in pretty much all cell lines, these chemical modifications improved the performance. This data shows HSPCs on the left and T cells on the right. In the publication, we also talk about K562, we've also heard about the performance -- we've also heard that these chemical modifications improved the performance of CHO cell lines. On the left side of this slide, it shows percent editing at 2 low sites, IL-2R gamma and HBB. For both low sites, an RNA, RNA approach was taken, that is, it was a single guide RNA, either unmodified methyl, MS or MSP, and then the Cas9 was delivered as mRNA. Both low sites showed that MS and MSP in-protection is necessary for exonuclease resistance. And thus, you see editing at about 15% to 20% with MSP and MSP modifications. The MS and MSP modifications provide more resistance to exonuclease than the unmodified or just the 2'-O-methyl group. The same is true for the image on the right side of the slide. In this case, T cells were edited at the CCR5 locus. Again, the MS and MSP in-protected guides provide resistance to exonucleases. They were more stable, and thus, you see a high level of editing at around 40% to 50%. Finally, on the far right of the image shows an RNP approach at CCR5. That is, the guide was delivered as synthetic RNA and the Cas9 was delivered as protein. The RNP provides some level of protection, likely due to the Cas9 enzyme complexing with RNA. However, we still see about a 2.5 fold improvement of editing efficiency when comparing MS in-protected guide compared to an unmodified synthetic guide. CRISPR is generating a lot of excitement for its potential as a therapeutic agent. We've seen some customers with experience in bioproduction getting into the cell and gene therapy space. There are labs and companies developing both ex vivo and in vivo therapeutic approaches for CRISPR. For ex vivo, the cells are removed from the patient, edited with CRISPR/Cas and then reintroduced back into the patient. And there's been a lot of work in this area, specifically around CAR-T and rare genetic diseases, such as sickle cell and beta thal. For in vivo editing, AAV is commonly used. There is direct editing of cells in the patient's body and a couple of examples include liver and ocular diseases. The reason why I'm mentioning this is because there's a division at Agilent that focuses on manufacturing DNA and RNA oligos under GMP conditions. And GMP-grade RNA oligos are used for the ex vivo delivery applications listed at the top. Here's a look at the site located in Boulder, Colorado on the upper right. We just opened a new site in Frederick, Colorado to keep up with this expanding market for GMP-grade oligonucleotides. Agilent's division is called NASD, or Nucleic Acid Solutions Division, and they are experts in large-scale synthesis under GMP conditions. They have made guide RNA at 100 nucleotide length at a several gram scale. They've also been audited by the FDA multiple times. And finally, this group has been making GMP-grade oligos for over 13 years. To summarize, Agilent offers custom end-to-end solutions for synthetic RNA with HPLC purification from research-grade to GMP-grade. On the left side, it shows our research-grade RNA, which is small scale, typically 100 micrograms up about 100 mg of material. We make custom guide RNA that can be paired with your favorite enzyme of choice, whether it's Cas9 or Cas12a or whatever variant you're working with. As I mentioned, we make long RNA, up to 164 nucleotides, with all sorts of chemical modifications to enhance the stability and specificity. Finally, looking on the right, it shows our GMP capabilities for large-scale synthesis. Collectively, Agilent's synthetic RNA offering spans from discovery through commercial stage. Next, we'll discuss our CRISPR library screen. The CRISPR library screens are used as a discovery tool. They're widely used in T cell research, and there have also been some recent examples in CHO cell library screens for bioproduction. CRISPR screens are powerful forward genetic screens, which allow you to look at hundreds or thousands of CRISPR engineering events in one assay. These screens can be gene knockout, gene activation or gene interference. The results yielded the discovery of new engineering targets. For bioproduction, CRISPR screens still look at the entire genome or gene subset such as metabolism, glycosylation or secretion. There was just an interesting CRISPR screen that was published by Nate Lewis and his colleagues on CHO cell metabolism. This screen resulted in the identification of a novel target, Abhd11, which when deleted substantially increased growth in glutamine-free media. Next, let's look at the CRISPR screening process in more detail. There are 4 key technologies which enable CRISPR-pooled screens. One, CRISPR/Cas genome modification, and as I've mentioned, that could be knockout, activation or interference; the second is pooled oligo synthesis; the third is lentiviral delivery; and fourth is deep sequencing, or NGS. The pooled library starts with the design of a guide RNA panel. So this could either be a genome-wide panel with like more than 100,000 different guides or, for example, a pathway-specific panel with only a couple hundred or a couple thousand guides. The guides are synthesized by our inkjet DNA printing technology and all oligos are printed simultaneously, and up to 244,000 different oligos can be printed at the same time. In mammalian cells, the guides are typically delivered by viral transduction, which requires the DNA oligo to be PCR-amplified, coned into a lentiviral vector, followed by packaging into viral particles. Once the viral particles are generated, they are introduced to the pool of cells at a concentration of about 1 guide per cell. Then the screen is performed and the guides are either enriched or depleted. This could be a growth screen, for example, and the publication showed the CHO cell growth in media without glutamine. The enriched or depleted cells are then deep sequenced for the correct guides and the hits are determined by the abundance of the guide construct, which correlates to the modification of a specific gene. After the screen is performed, we can sequence the cell to determine which gene knockouts helped grow this glutamine-free media. Let's look at pools -- pooled oligo synthesis in more detail. Agilent's inkjet DNA printing technology simultaneously prints single stranded DNA in high complexity parallel process. We can make up to 244,000 different oligos up to 230 nucleotides in length, the DNA is printed and then cleaved off. For CRISPR libraries, we PCR-amplify the single-stranded DNA and clone them into lentiviral plasmid and finally package into lentiviral particles. Lentiviral delivery is one of the key technologies for CRISPR libraries. Lentivirus is ideal because it integrates into the genome. The goal with this delivery is to get 1 lentiviral guide per cell, and this will allow us to correlate the phenotype to this -- to the guide, and thus, the gene that we targeted for either knockout, activation or interference. The bottom of this slide shows Agilent's CRISPR library offering. For bioproduction, these guides will likely be customed. CHO cells, for example, would be a custom print, followed by PCR cloning and lentiviral packaging. Next, let's discuss the oligonucleotide synthesis library, or OLS, by inkjet printing technology in more detail. Specifically, let's dive into DNA oligo sequence fidelity, representation and customization. Agilent synthesis leads to high fidelity oligos with our standard process with an error rate of 1 in 300. We are implementing a high fidelity synthesis process that has an error rate of better than 1 in 1,000. So as expected, the oligos that we intend to make are correct. Representation is the idea that each guide is present at a 1:1:1 equimolar ratio. And to assess representation, we have tested internal libraries of oligos by deep sequencing the library, counting the number of reads for each unique oligo sequence and then normalizing this to the average we see across the pool. If there was a publicly uniform distribution, we would see a single spike at 1 in the middle top part of that image, with every oligo with an exact equimolar proportion to every other with that spike at 1. Of course, in reality, we do see a distribution, and the tightness of that distribution of sequence read shows the library has an excellent distribution. Our goal is to have oligos in the 90th percentile at no more than a threefold higher level than those in the 10th percentile, and this is referred to as our 90/10 ratio. Libraries with longer tails, the red curve, for example, will have a large number of overexpressed and underexpressed sequences, which can lead to false positives and false negatives in your screen. The internal testing of Agilent library showed that 60,000 oligos routinely only found 1 missing sequence and 90/10 ratios of less than 3. This data is seen at the -- in the table at the bottom of the image. Representation is important for CRISPR screens because you want to ensure that you have all the guides present at the time of your screen. During synthesis and cloning, guides can be lost or enriched. Therefore, it's important to monitor representation by NGS. Since all guides are delivered as lenti and only differ in their 20 nucleotide CRISPR sequence, we can easily sequence and count the reads. In addition to high fidelity DNA synthesis, we also developed an optimized cloning system to help with representation. This is an improved version of Gibson Assembly, which uses an overlap assembly based -- an overlap-based assembly for integration. There are no restriction digest as it works like a flap mechanism. Short vector provides a more stringent molecular selection for correctly assembled constructs compared to Gibson. And this results in about a 15% to 20% improvement in the quality of your plasmid libraries that are cloned in your lab. And here's the data looking at an OLS print, a Gibson cloning reaction and a SureVector cloning reaction. If you look on the left side, we can see 3 traces. Orange is the OLS print. There are 0 missed guides. It's a very tight distribution. Gibson is in green, and the width of the bell curve is larger than -- is larger with Gibson compared to SureVector or OLS. And this is showing less representation. More guides are overrepresented and also underrepresented. SureVector is in blue, and it only missed 7 guides and had an optimal 90/10 ratio of 2.5. Therefore, SureVector cloning is an improvement over Gibson. When you generate a pooled CRISPR library, you do the cloning reaction and amplify the library in E. coli. Agilent developed a 3D growth matrix to help the representation during the E. coli amplification step. The 3D growth matrix provides the surface area similar to a liquid broth and the growth uniformity of plates. Essentially, the 3D matrix combines the best features of the liquid broth as well as with a plate. So when it comes time to harvest your 3D gel, you simply shake the bottle after they grow for 2 days or so, you pour into a centrifuge bottle, you spin down and harvest your clone. After isolating the plasmid library, the clones are ready for lentiviral packaging. Finally, the third component of Agilent's printing technology is customization. As I mentioned, you can have up to 244,000 different oligos printed at the same time, and this can be up to 230 nucleotides in length. We also have catalog libraries for knockout, activation, interference in human and mouse and custom DNA prints for all other species, including CHO. So now you have a high-quality CRISPR library. What now? Well, at that point, you can do a number of things in terms of performing your screen, whether it's a genome-wide screen or a pathway-focused screen looking at a few hundred to over 200,000 different guides. We also need to consider the experimental design. What cell line we're going to test, the number of cells. Many times the number of cells can be quite high in these screens. How many replicates we're going to perform? What sort of selection we're going to do? And if we've got an NGS protocol and analysis pipeline? There are several NGS software packages out there for CRISPR library screens to look at the data. Okay. So the final technology to CRISPR screens is NGS. And just to quickly recap the workflow, we print the oligos, we clone them. We express them as lenti. We introduce the lenti and perform cell line selection. And the lentiviral insertion really access the bar code here, and we can follow the bar code with NGS. We also want to perform the screen with and without Cas9. And finally, we want to run next-gen sequencing on both the no Cas9 and the positive Cas9 experiment. And this experiment should be run in duplicate and preferably, triplicate. Since the lentiviral vectors only differ in their 20-nucleotide CRISPR sequence, we can PCR this section throughout the workflow to ensure representation is maintained. After the screen is performed, NGS is used to count the guide frequency, and the data is displayed comparing the guide frequency before selection to guide frequency after selection. And this is shown in the bottom right image. As you design your screen to contain 4 to 10 guides per gene, you should see genes of interest change in accordance with each other. CRISPR screens are typically pretty clean and the different guides should correlate nicely. Once the hits are identified by NGS, these hits can be further validated with cell line engineering, where you modify one gene at a time. So for example, you could use like a synthetic guide that we talked about in the first half of the talk with a purified Cas9 enzyme. Other important things that we should consider when looking at the data is that the no Cas9 control, the library shouldn't show too much bias towards any one CRISPR. All the libraries will have some sort of background or noise or bias, but it's -- with the Cas9, the library should start to show some bias towards certain CRISPRs due to the selection or whatever screening you're performing. However, a bad library will have more noise, and it will make it harder to identify the significant CRISPRs. This requires your screen to encompass a greater number of cells. This is the summary slide of what we've been talking about regarding catalog and custom libraries. For bioproduction, these will likely be custom CHO cells, for example, and those would be a custom OLS print on the far right. Some of the human libraries may be useful if you're working in HEK-293, and these are catalog libraries shown on the left. The whole genome knockout is the GeCKO v2, and we also have CRISPR activators and interference that was licensed from the Weissman Lab. To conclude, we just discussed a couple of Agilent technologies for CRISPR/Cas genome engineering, our synthetic RNA via our TC-RNA chemistry and our inkjet-printed DNA for CRISPR libraries. As many know Agilent for its world-leading analytical tools such as LC/MS, we are also committed to bringing high-quality products in the genomics and molecular biology space. I would like to thank you for your time and attention, and I will gladly answer any questions, and I'm happy to discuss with you any of your CRISPR project goals. So with that, I will pass it back to Eden for time for Q&A.

Thank you very much, Dan. That was Dan Fisher, from Agilent. Thank you for that excellent presentation. So we've received a few questions already, but we're just going to give the rest of you a moment to enter your questions in the Q&A box to the left of the slide. So just before we begin the Q&A, I've just got a few brief announcements for everybody. So I'd like to thank Agilent for sponsoring this digital week. I'd just like to draw your attention quickly to our face-to-face BioProcess International events visiting Boston in September and Milan in October this year. Additionally, BioProcess International Europe will be delivered as a 100% virtual conference and exhibition on July 13 to 17 this year. Also be sure to check out the Resources List to the right of your screen, where you can download a few featured white papers. So now we're going to go back to Dan and have a look at some of the questions we've received. So do you suspect your chemically synthesized sgRNA will help CHO cell editing, i.e., CHO cells do HR poorly?

Yes, that's a good question. Yes. I think for most knockout applications like NHEJ, the chemical modified guide will work pretty well within CHO cell editing. One of the challenges with CHO cells is that they do HR pretty poorly. So these modifications, in theory, should help because they're going to be more stable, they're going to hang around longer, you're going to get better editing. But since it does HR pretty poorly, those percentages may be still pretty low. I did mention a little bit about prime editing, which is a new technique that allows you to integrate without a double-strand break. But since that information is contained within the guide, the size of that insert can be limited. So yes, I think that these chemical modifications would give us our best shot at CHO cell editing.

Fantastic. I've got another one here. If I want to knockout a contaminating host cell protein in my clone, what's the best approach for testing this?

Okay. Yes. So if you're going to knockout a protein, one common way with CRISPR/Cas9 is that you can essentially set up several different guides that can be kind of tiled along that gene. And then you could effectively screen 2, 3, 5 or even 10 or 20 different guides for that gene. And then you would deliver those one at a time and then test the editing efficiency. But also not only the editing efficiency but also the protein level of whatever contaminant that you're trying to knockout because you're going to want to check -- essentially, you're going to want to get the guide that has the highest editing efficiency and also knocking out the contaminant protein at the highest efficiency as well.

Fantastic. And another one that's just come in is, what percentage of purity is your gRNA?

Yes. Our purity is, like, for a standard 100 mer is I mentioned 80% in the talk, but we typically see over 90% for 100 mer. And then I think some of the data that we've seen for, like, the 130 mers, 140 mers, our spec is over 80%. So we're always going to give guides that are over 80%. Sometimes they can go over 90%, depending on the size. If they become too long, then they'll probably stay at over 80%.

That's great. And we typically use CRISPR/Cas9 plasmids and/or IVT guides. What are the advantages of using CRISPR/Cas9 in RNP format?

Yes. So we touched on this a little bit. So IVT guides are good, but the problem is that some of the IVT guides -- some of the polymerases that turn that plasmid into the RNA require that you start with a G, which is not going to be the case with chemical synthesis. You can just start with whatever nucleotide that you want. There's also more consistent manufacturing and scalability with these chemical synthesis methods. And then also the placement of chemical modifications. So an in vitro-transcribed guide is just going to be copied off the DNA template, right? So you can't incorporate chemical modifications with IVT. But with chemical synthesis, we can just precisely play like a 2'-O-methyl-3'phosphorothioate on the 5' and 3' ends, adding stability. So you can do -- you have more flexibility with the chemical synthesis method, I would say.

That's great. And we've got another one. As your chemically modified gRNA, could we produce your amidites as second supplier if we need more gRNA than your capability?

I'm sorry. Could you repeat that question? I kind of missed that.

Yes. Sorry, maybe I said it wrong. So as your chemically modified gRNA, could we produce your -- amidites as second supplier if we need more gRNA than your capability?

Okay. Let me see if I can find. Yes. Let me see if I can -- I think I found that question. I'm looking at it right now. Yes, we probably have to take that one -- let me take that one offline. I'm not really sure about the supply chain and what that looks like. Yes, we probably should take that one offline. If you could -- yes, I think you could e-mail me. My e-mail is [email protected]. I'd be happy to follow-up with you on that specific question.

That's great. So let's get some of the others. So what is the longest synthetic RNA that you can make?

Well, commercially, we do 164. And the reason why we do 164 is just because of that CRISPR activator system. We had people that wanted to make that long of a guide. So commercially up to 164, but we're trying longer than that at the moment. Like, with specific -- like, with these prime editing applications that require longer guides, they can be -- they're typically about 125 and they can be 200 or longer depending on what the desired modification is. So 164 commercially, but we're actively pushing that higher.

That's great. And if I only want to look at a certain pathway for a CRISPR screen, what's the best path forward?

Okay. Well, if you're only concerned with a specific pathway, I think the first question would be, like, what cell line you're using. So CHO is the Chinese Hamster, right? So it's not like a standard human or mouse. So if you're doing CHO work, it's -- we would need to just print that and then go from there. But if it was -- if you're doing like HEK-293 and if you could use human -- we do have full genome human libraries as well as subsets like kinase and phosphatase and those sort of subsets as well. So I think depending on what cell line, what species you're working in will probably kind of dictate which way you want to go. If you want to -- for CHO, I would recommend just printing them from scratch. And then if it's a human or mouse, we likely have an off-the-shelf solution for you.

Fantastic. And one that's just come is, have you made a CRISPR knock in whole genome library?

Did you say knock in or knockout?

knock in, yes.

We haven't done the knock in yet. I think I was just reading a recent publication on that, but we have not -- we don't have that available for knock in, just knockout.

And we have, could you add 5' capping and 3' poly (A) to the synthesized sgRNA?

That's a good question. So those are -- so the capping and the 3' tail is common for, like, a messenger RNA, right? With the chemical synthesis, it starts with a solid support. Many times it's glass, it can be something else. And then we basically are adding beads to that string or nucleotides to that string. Let's see here. I don't -- I think it will depend -- I wonder what sort of cap that they're looking for. We do all sorts of custom chemical modifications. So that may be one that I would need to follow-up with our scientists to see. I don't know about that 5' cap. The 3' poly (A) tail, we can -- since we're doing chemical synthesis, we can add as many As as we want on the end. So we can definitely do that. But it is a little different than -- so these synthetic guides are just a little different than like a normal mRNA. But depending on what the application is and what the goals are, there may be some solutions for you.

That's great. Another one that's just come in is, how important is the purity of gRNA?

Yes, a good question. When I think of pure -- I mean, obviously, the more pure the better. And especially if we think about how CRISPR works, right, so you remember -- so the first 20 nucleotides on that CRISPR RNA drive the specificity. So if we do have impurities, and depending on where those impurities lie within the guide, we certainly have opportunities for more off-target cleavage. One of the common impurities is an n-1 oligo. So, like, if you're working with the normal 100 mer, the #1 contaminant would be like an n-1 or a 99 mer. And then another contaminant would be like an n-2. So ideally, you would like to weed away those impurities if possible just for the cleanest gene editing. Now in practice, I think it will just come down to how well do your guides work for your specific application. Now if you're getting, like, 70%, 80%, 90% editing with whatever you're using now, then you certainly don't need to change that, right? And then I think the second part of that question is, is it insufficient produce but -- yes, so they're asking about TBDMS, amidites and -- Yes. And so I think, ultimately, it's just going to depend on your application. And because certainly, TBDMS is a widely used chemistry and a lot of those guides work just fine. So I think it's -- if you're having issues with your current approach, that you may want to consider this TC-RNA chemistry. But if whatever you're working with is working fine, then I wouldn't recommend changing it.

Great. Well, thank you so much, Dan. So that is all the time we have for questions today. Thank you so much for a great session. And if anyone submitted a question that wasn't addressed, keep in mind the speaker will reach out to you directly. This session was recorded. You will see the notification in 24 hours and the on-demand session is available for viewing. Before you all log off, please take a moment to complete the feedback form, so we can continue to improve your Digital Week experience. I hope you enjoyed today's session. And on behalf of Informa Connect Life Sciences, I hope you enjoy the rest of your day. Thank you very much.