High Purity Guide RNA for CRISPR Screening Applications

trilinkbiotech.com | 1 Highly pure, reproducible guide RNA screening via ligationbased synthesis platform White paper Features • The critical role of gRNA purity in high-throughput screening: CRISPR success depends on selecting gRNAs with high specificity and editing efficiency. Inconsistent gRNA purity undermines reproducibility, making high-purity synthesis essential for reliable screening and confident candidate selection. • A new workflow for gRNA manufacturing: To address persistent challenges in gRNA manufacturing for high-throughput screening, we developed a ligationbased workflow that enhances purity and consistency. This approach minimizes synthesis-related variability, enabling more reliable and reproducible CRISPR results. • Experimental validation and supporting data: We evaluated our ligation-based workflow through experiments measuring reproducibility and sequence fidelity. The method consistently delivers gRNAs with purities above 80% and reliable yields, supporting uniform dosing for high-throughput applications. trilinkbiotech.com | 2 Introduction Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) geneediting technology is transforming the field of therapeutics, driving the development of both established and emerging treatment modalities. The therapeutic potential of CRISPR-based technologies lies in their ability to precisely modify the genome by introducing targeted edits at specific DNA sequences. This is achieved through a custom-designed guide RNA (gRNA) that directs a Cas enzyme, such as SpCas9 or SaCas9, to the desired site, where it creates a DNA break for repair and genetic modification. This can result in the correction of diseasecausing mutations, the regulation of gene expression, or the disruption of harmful genetic elements, offering new treatment strategies for a wide range of diseases 1. The effectiveness of a CRISPR-based therapeutic depends on gRNA design and quality, which determines the system’s specificity and efficiency 1. Thus, selecting an optimal gRNA is essential to ensure accurate editing and minimize off-target effects that could impact efficacy. The gRNA selection process requires evaluation of candidate gRNAs to identify those most likely to perform reliably in preclinical and clinical applications, a process guided by CRISPR screening 2. CRISPR screening involves systematically testing large numbers of guide RNAs in cells to measure their editing efficiency, specificity, and functional impact (Figure 1). This high-throughput screening can be used as a tool to identify gRNA lead candidates in cell and gene therapies and other applications 2. Considerations for gRNA quality in high-throughput screening High-throughput screening approaches have accelerated the identification of candidate gRNAs with therapeutic potential 2. However, despite their scalability and precision, these technologies remain vulnerable to technical limitations that can compromise data integrity. Issues of sensitivity and reproducibility are significant in largescale screens, where even minor sample-to-sample variation may produce false positives, reduce overall reliability, or result in false negatives 3. As the scale of CRISPR screening expands, maintaining sample integrity and minimizing experimental interference becomes critical for ensuring dependable outcomes. Mitigating these risks requires the use of high-purity gRNAs. In prioritizing speed and cost reduction, most commercial suppliers perform only minimal purification during plate production 3. As a result, these plate formats are prone to variability in yield and/or purity, making them unreliable for generating reproducible results. This additional variability in an already complex study may result in false positives or cause researchers to overlook promising gRNAs due to poor purity. To overcome these challenges and maintain data integrity and ensure dependable outcomes in high-throughput CRISPR screens, it is therefore essential to prioritize high-purity gRNAs. White paper Design and order gRNAs Transfect cells Culture and select Phenotype and assess results Figure 1: Overview of a high-throughput screening workflow designed to select candidate gRNAs for downstream validation while excluding non-functional sequences. trilinkbiotech.com | 3 Ligation-based workflow for enhanced gRNA manufacturing To address these challenges, we developed an innovative ligation-based synthesis platform that enhances gRNA quality for high-throughput screenings in an automated 96 well plate format (Figure 2). Two RNA components are synthesized separately and then ligated to form the full gRNA. A dedicated cleanup step removes truncated or remaining fragments, minimizing cross-contamination and reducing well-to-well variability. This new synthesis and purification platform results in gRNA with higher purity than standard desalting, enabling more reproducible screens and more accurate lead identification for downstream CRISPR applications (Figure 3). Compared to desalted gRNA, which shows lower and more variable yields well-to-well, our new ligation-based method delivers higher yields and more consistent yields of full-length product (Figure 4). Data To assess the quality and strength of our innovative workflow, we performed a variety of experiments to assess the reproducibility and sequence robustness. We initially started by refining our process conditions and comparing our results to a typical desalted gRNA to assess overall purity (Figure 3). From there, we gradually increased the total number of sequences produced to perform >200 ligation reactions and assessed both the yield and purity of the resulting material to ensure the method was scalable for high throughput production (Figure 4 & 5). We have shown that our method is able to consistently produce gRNAs at purities >80% with reliable yields for uniform dosing, making our solution fit for custom high-throughput screening. Figure 3: Comparison of standard desalted gRNA versus ligation-produced gRNA. Both gRNAs are 100-nucleotide SpCas9-matched sequences analyzed using our standard quality and release assay. Desalt gRNA had a measured purity of 25% while the gRNA produced by ligation was >70% pure. Figure 2: Our high-throughput ligation workflow builds full-length gRNA from two RNA components, with a post-ligation cleanup that removes residuals and truncations for consistently high purity across every well. Desalt gRNA Truncations Truncations Longmers Longmers Desired product Desired product Ligated gRNA Protospacer sgRNA scaffold Ligated sgRNA Ligation Clean up Unligated materials Final full-length sgRNA 10770 Wateridge Circle, Suite 200 San Diego, CA 92121 | 800.863.6801 | trilinkbiotech.com Please note that our discovery services are for research use only. Not for use in diagnostic or therapeutic procedures. Not for use in humans. © 2025 TriLink BioTechnologies. v.09122025 Conclusion Our ligation-based synthesis platform significantly elevates the quality and reliability of high-throughput screening workflows. By ensuring high-purity and batch-to-batch consistency of gRNA, it enables robust reproducibility across experimental replicates and study conditions. We are actively expanding this technology to support a broader range of genome editing systems, with the goal of making high-fidelity screening accessible across diverse research applications. Ready to take your screening workflow to the next level? For more info, visit: trilinkbiotech.com/screening-gRNAs Interested in collaborating with us? Let us know: maravai.com/collaborate References 1. Li, Tianxiang, et al. “CRISPR/Cas9 Therapeutics: Progress and Prospects.” Nature News, Nature Publishing Group, 16 Jan. 2023, www. nature.com/articles/s41392-023-01309-7. 2. Zhang, Heng, et al. “Deep Sampling of Grna in the Human Genome and Deep-Learning-Informed Prediction of Grna Activities.” Nature News, Nature Publishing Group, 16 May 3. Zhang, Yuchen, et al. “High-Throughput Screening for Optimizing Adoptive T Cell Therapies - Experimental Hematology & Oncology.” BioMed Central, BioMed Central, 13 Nov. 2024, ehoonline.biomedcentral.com/articles/10.1186/ s40164-024-00580-w. SpCas9 (n=96) SaCas9 3 4 2 1 0 Figure 5: Representative yield results for 96-well plate production of SpCas9 and SaCas9 gRNAs. Average yield values were 2.5 nmol for SpCas9 and 2.9 nmol for SaCas9. SaCas9. Yield of full-length product was determined by the following equation: rYield FLP = ((ODs/ε) • Purity(%)) • 1000 Figure 4: Representative purity results for 96-well plate production of SpCas9 and SaCas9 gRNAs. Purity was determined using our standard analytical method and verified by independent review. Average purity values were >80% for SpCas9 and >65% for SaCas9. Desalt purity (%FLP) Desalt FLP yield