Empowering CRISPR With Innovative Single-Cell Isolation

Empower CRISPR Cell Engineering with Innovative Cell Sorting & Single Cell Dispensing CRISPR Introduction In biomedicine, one of the most revolutionizing tools is the CRISPR/Cas9 system, which allows modification of sequences in the genome with precision and efficiency. CRISPR/Cas9 can be used with immortalized cell lines, primary cells, and induced pluripotent stem cells (iPSCs) to build models for studying gene functions, disease mechanisms and therapeutic approaches. When combined with induced pluripotent stem cells (iPSCs), CRISPR/Cas9 is especially powerful. It facilitates the precise editing of iPSC genomes, creating models that mirror disease-specific genetic aberrations1 . These models are invaluable for delving into disease mechanisms and pioneering new treatments. Furthermore, by rectifying genetic anomalies in iPSCs, scientists can generate cells tailored to a specific immune profile, paving ways for personalized cell therapies for a host of diseases2. 02 Single Cell Isolation in CRISPR Cell Engineering While CRISPR technology brings a new paradigm in cell engineering, it is not without challenges. Off-target editing and variable editing efficiency makes it critical to isolate clonal populations to further characterize the edited cells to identify clonal cells with desired characteristics. Working with clonal populations ensures that each clonal line of cells being studied possess heterogeneous genomic makeup and consistent biological behavior. Renowned for its fast, gentle and easy platform, Bio-Techne’s 2-laser Pala Cell Sorter and Single Cell Dispenser enhances the speed and efficiency that are crucial for the next wave of cell engineering breakthroughs. Learn more about the Bio-Techne Pala Cell Sorter & Single Cell Dispenser bio-techne.com/instruments/single-cell-dispensers BIO-TECHNE ® // EMPOWER CRISPR 03 "The Bio-Techne Pala Platform has been increasingly recognized by leading scientists & researchers..." The Bio-Techne Pala Cell Sorter and Single Cell Dispenser platform has been increasingly recognized by leading scientists and researchers as the tool of choice for gently dispensing single cells to generate clonal cells after gene editing, with or without fluorescent tagging. Combining microfluidics, flow cytometry and liquid dispensing, the Pala platform, with its remarkable ease-of-use, is serving research groups of all sizes and helping scientists get to their optimal clones more efficiently, as highlighted by the following publication briefs. Featured in PeerReviewed Citations Hypoimmune Induced Pluripotent Stem Cells Survive Long Term in Fully Immunocompetent, Allogeneic Rhesus Macaques Published in: Nature Biotechnology Rejection of cell therapeutics by the immune system is a critical aspect taken into consideration during cell engineering and cell therapy development. The study in this publication demonstrates how hypoimmune pluripotent (HIP) stem cells were engineered and transplanted into allogeneic rhesus macaques. The HIP cells successfully survived in these macaques and differentiated into various lineages. Human HIP cells were also studied, where they were differentiated into pancreatic islet cells, resulting in measurable improvements in diabetic mice. A Bio-Techne Cell Sorter and Single Cell Dispenser was used to generate the foundational HIPs for the study. It facilitated two rounds of precise single cell cloning to create B2M-/ -CIITA-/ - iPSCs via CRISPR gene inactivation. This process involved depleting MHC class I & II expressions. The gentle cell handling by the Bio-Techne platform ensured cell viability. 01 0 4 Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin & Histone H1 Published in: Biomolecules This research aimed to enhance high-throughput cancer drug screening by utilizing a cell line model with a GFP tag added to the β-tubulin gene through CRISPR. By inhibiting β-tubulin polymerization, one can gauge the restriction of proliferation. The GFP tag facilitates real-time monitoring of β-tubulin polymerization via high-content imaging. This live-cell approach expedites the screening process and boosts accuracy since it excludes additional cell preparation and allows genes to be observed in their most physiological state, thus offering a truer assessment of drug impacts. In the study, the Bio-Techne Cell Sorter and Single Cell Dispenser played an integral role by efficiently dispensing single cells for cloning after CRISPR editing. This optimization by the Bio-Techne platform streamlined the cloning process, resulting in a larger number of viable clones. These clones were pivotal for screening desired traits like precise editing, minimal off-target effects, and robust proliferation. Redefining the Role of Ampk in Autophagy & the Energy Stress Response Published in: Nature Communications This study revisits the role of adenosine monophosphateactivated protein kinase (AMPK) in autophagy, challenging the current understanding. It uncovers AMPK’s dual functions: moderating the immediate induction of autophagy during energy deficits while preserving vital autophagy components. These dual roles are essential for cellular balance and survival during energy stress. The Bio-Techne Cell Sorter and Single Cell Dispenser played a pivotal role in creating various knockout (KO) and double knockout (DKO) cell lines postCRISPR editing. GFP-positive cells underwent sorting and were then dispensed for using the Bio-Techne platform, establishing distinct clonal lines. The KO cell lines were instrumental in shedding light on the functions of the genes that were edited out. CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease Published in: Antioxidants Non-alcoholic fatty liver disease (NAFLD) impacts 20–25% of the global populace, a figure that intensifies with the growing prevalence of obesity while therapeutic solutions are scarce. To address this, the research team utilized an engineered cell line model to perform high-throughput screening on a vast library of FDA-approved substances. They identified six compounds with potential as therapeutic agents for NAFLD. The Bio-Techne Cell Sorter and Single Cell Dispenser played a crucial role in the research methodology. Utilizing CRISPR, the team tagged the endogenous heme oxygenase-1 (HMOX1) gene with a luciferase reporter in HEK293T cells. The Bio-Techne platform then dispensed the edited cells for cloning. The accurately edited clone served as the foundational model for the subsequent drug compound screening. 02 03 04 BIO-TECHNE ® // EMPOWER CRISPR 05 CRISPR-Directed Gene Editing as a Method to Reduce Chemoresistance in Lung Cancer Cells Published in: Springer Link This research introduces an innovative approach to improve cancer treatment outcomes by curtailing chemoresistance. By targeting genes believed to have acquired resistance during chemotherapy, radiation, or immunotherapies, the strategy involves using CRISPR editing to introduce mutations that incapacitate the specific gene. In a practical application of this concept, the team worked with a lung cancer cell line, editing the NRF2 gene to integrate two mutations. The Bio-Techne cell sorter and single cell dispenser was instrumental in this endeavor. Over two rounds of editing and cloning to develop the desired cell line, the Bio-Techne Platform efficiently dispensed individual cells, streamlining the meticulous process of generating the mutation-bearing lung cancer cell line. Age-Related Low Bone Mineral Density in C57BL/6 Mice Is Reflective of Aberrant Bone Morphogenetic Protein-2 Signaling Observed in Human Patients Diagnosed with Osteoporosis Published in: International Journal of Molecular Sciences Osteoporosis is a prevalent age-linked bone ailment. While potential treatments, BMP-2 and CK2.3, operate via the BMP-signaling pathway, their limitations underscore the need for dependable mouse models to understand associated complications. Drawing insights from a cell line model where the BMPR1a receptor (integral to BMP-2) was nullified using CRISPR, this study successfully showcased the C57BL/6 mouse strain as a reliable model for osteoporosis research. The pivotal knockout cell line, foundational to this research, was crafted using the capabilities of the Bio-Techne cell sorter and single cell dispenser. Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phosphoregulation, HIV-1 restriction & cellular dNTP levels Published in: bioRxiv This paper holds significance for the scientific community due to its pioneering approach in studying HIV-1 infection within human macrophages. By introducing mutations to the SAMHD1 catalytic core locus in BLaER1 cells, the research offers a more physiologically relevant model for examining SAMHD1’s anti-viral functions. This stands in contrast to the conventional methods that rely on SAMHD1 over-expression, which may not truly replicate in vivo conditions. The novel approach paves the way for devising strategies that target SAMHD1 with precision, without unintended interference in other cellular operations. The successful creation of both knock-out and knock-in cell lines, central to this study’s innovations, was made possible through the utilization of a Bio-Techne cell sorter and single cell dispenser, further underlining its importance in advancing cellular research methodologies. 05 06 07 06 BIO-TECHNE ® // EMPOWER CRISPR 07 Trusted by Leading Institutions The researchers who published the citations featured in this monograph chose Bio-Techne Cell Sorters and Single Cell Dispensers for their cell engineering workflows due to the ease-of-use and gentle sorting that preservers cell viability. Fast Bio-Techne’s Pala cell sorting and single cell dispensing platform drastically reduces instrument overhead time, letting you go from initializing the instrument to achieving plated single cells in as little as seven minutes. This is in contrast to conventional FACS systems, which can take over an hour to operate and provide results. Easy The Pala platform is automated and easy-to-use. The plug-and-play user interface streamlines many of the steps required for traditional FACS sorters, saving scientists and researchers valuable time and money. The simplicity of the platform makes it possible for anyone to use without specialized expertise or training. Gentle At less than 2 psi, the Bio-Techne Pala Cell Sorter and Single Cell Dispenser uses low-pressure sorting to accurately and efficiently sort cells gently, thus preserving cell viability and integrity for better clonal growth. 08 BIO-TECHNE ® // EMPOWER CRISPR 09 10 1. Hu, X., White, K., Olroyd, A.G. et al. Hypoimmune induced pluripotent stem cells survive long term in fully immunocompetent, allogeneic rhesus macaques. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023- 01784-x 2. Park, JM., Lee, DH. & Kim, DH. Redefining the role of AMPK in autophagy and the energy stress response. Nat Commun 14, 2994 (2023). https://doi.org/10.1038/s41467-023-38401-z 3. Li, J., Arest, S., Olszowy, B., Gordon, J., Barrero, C. A., & Perez-Leal, O. (2023). CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease. Antioxidants (Basel, Switzerland), 12(7), 1363. https://doi.org/10.3390/antiox12071363 4. Khachatryan, H., Olszowy, B., Barrero, C. A., Gordon, J., & Perez-Leal, O. (2023). Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1. Biomolecules, 13(2), 249. https://doi.org/10.3390/ biom13020249 5. Rivera-Torres, N., Bialk, P., & Kmiec, E. B. (2023). CRISPRDirected Gene Editing as a Method to Reduce Chemoresistance in Lung Cancer Cells. Methods in molecular biology (Clifton, N.J.), 2660, 263–271. https://doi.org/10.1007/978-1-0716- 3163-8_18 6. Halloran, D., Pandit, V., MacMurray, C., Stone, V., DeGeorge, K., Eskander, M., Root, D., McTague, S., Pelkey, H., & Nohe, A. (2022). Age-Related Low Bone Mineral Density in C57BL/6 Mice Is Reflective of Aberrant Bone Morphogenetic Protein-2 Signaling Observed in Human Patients Diagnosed with Osteoporosis. International journal of molecular sciences, 23(19), 11205. https://doi.org/10.3390/ijms231911205 7. Schüssler, M., Schott, K., Fuchs, N. V., Oo, A., Zahadi, M., Rauch, P., Kim, B., & König, R. (2023). Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV1 restriction and cellular dNTP levels. bioRxiv: the preprint server for biology, 2023.08.24.554731. https://doi. org/10.1101/2023.08.24.554731 8. Paolini Sguazzi, G., Muto, V., Tartaglia, M., Bertini, E., & Compagnucci, C. (2021). Induced Pluripotent Stem Cells (iPSCs) and Gene Therapy: A New Era for the Treatment of Neurological Diseases. International journal of molecular sciences, 22(24), 13674. https://doi.org/10.3390/ ijms222413674 9. Chun, Y. S., Byun, K., & Lee, B. (2011). Induced pluripotent stem cells and personalized medicine: current progress and future perspectives. Anatomy & cell biology, 44(4), 245–255. https://doi.org/10.5115/acb.2011.44.4.245 References BIO-TECHNE ® // EMPOWER CRISPR 11 6460131923 Global info@bio-techne.com, bio-techne.com/find-us/distributors North America TEL 800 343 7475 Europe // Middle East // Africa TEL +44 (0)1235 529449 China info.cn@bio-techne.com, TEL 400.821.3475 For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners. Contact Us Global Developer, Manufacturer, and Supplier of High-Quality Reagents, Analytical Instruments, and Precision Diagnostics. INCLUDES R&D Systems™ Novus Biologicals™ Tocris Bioscience™ ProteinSimple™ ACD™ ExosomeDx™ Asuragen® Lunaphore™