Olivia Hughes from Sphere Fluidics discusses how advances in picodroplet microfluidics, combined with the high efficiency of CRISPR/Cas9 technology, are fuelling a new wave of innovation in the form of a simple click-through workflow for genome editing.
Picodroplet microfluidics is rapidly becoming the technology of choice for high-throughput single cell analysis, providing an easy-to-use tool to isolate single cells in picolitre-volume, water-in-oil droplets (picodroplets). Within these miniature ‘test tubes’, single cells can be assayed and screened with increased throughput, exceptional sensitivity, and reduced reagent volume.
Across industry and academia, these high-throughput microfluidic platforms have proven transformative in antibody discovery and cell line development workflows. Now, researchers have successfully applied picodroplet technology to applications such as genome editing and cell therapy to streamline the screening, sorting, and enrichment of desired clones.
Current challenges in the genome-editing workflow
Genome editing is a fast-growing market projected to reach $11.2 billion in 2025, at a CAGR of 17%1. The rising demand and number of clinical studies for cell-based therapies for the treatment of cancer and other genetic disorders are significant factors propelling this growth. Within this field, the high efficiency of CRISPR/Cas9 technology, a powerful form of genetic engineering, has transformed the targeting range and precision with which researchers can modify genomes. Yet, the production and testing of genome-edited cell lines remain complex and inefficient2.
The genome editing of mammalian cells typically begins with the delivery of DNA, RNA, and the Cas9-gRNA complex inside the cells, generating a mixed pool of edited cells. In a conventional workflow, common gene delivery approaches include electroporation, viral transduction, and lipid-mediated transfection. Next, to transition from a heterogeneous cell pool to a single cell clone, researchers conduct single cell isolation by limiting dilution or flow sorting, followed by the downstream analysis and validation of clones using DNA, RNA, and protein analysis techniques such as next-generation sequencing and microscopy methods.
The isolation and expansion of individual clones with the desired modifications and subsequently screening the desired phenotypic changes for each clone remain major hurdles in genome editing. The workflow depends on multiple tools and manually intensive steps requiring additional wet lab work, resulting in a complex and time-consuming workflow that can take several months to complete. To fulfil the potential of the technology, developments that consolidate steps in the gene-editing workflow to establish efficient and streamlined processes are significant areas of focus.
Picodroplet microfluidics for high-throughput screening and single-cell isolation
Utilising the combined benefits of picodroplet-based microfluidics and automation, researchers have achieved promising advancements to miniaturise and streamline the genetic engineering workflow. The potential of the technology has been demonstrated in various studies for genome editing, highlighting industry progress towards the objective of creating an automated platform for the delivery, sorting, selection, validation, and expansion of single edited clones.
Picodroplets act as miniature reaction chambers, enabling the compartmentalisation of single cells in a cell-friendly microscale environment, in which cells can be cultured for prolonged periods (Figure 1)2. The miniaturisation to picodroplet volumes offers several advantages over conventional processes, including dramatically reduced reagent costs and increased detection sensitivity due to the higher concentration of key molecules trapped in a picodroplet3. Such miniaturisation helps to improve the delivery of gene-editing machinery due to the close proximity of cells and reagents. The picodroplet format also enables the easier detection of factors expressed by single cells following a successful editing event.
These innovative technologies are primed for integration and automation. Advanced solutions comprise liquid handling, high-speed imaging, light microscopy, laser detection, and sorting, providing a single device to streamline workflows and reduce laborious and time-consuming processes for researchers3. Breakthrough commercial solutions pair the high-throughput instrumentation with intuitive, AI-driven single cell analysis software for data processing, analysis, and storage, representing another step towards a fully integrated, gene-editing system.
Automating the creation of genome-edited cell lines — from transfection to validation of the edited cell
The feasibility of using picodroplets in cell engineering and gene editing applications has been increasingly demonstrated in the literature2,4,5. In a study illustrating the entire mammalian genetic engineering pipeline in picodroplets, researchers begin by reinforcing the compatibility of picodroplets with a variety of widely used transfection reagents, including liposome-cationic lipids, non-liposomal lipids, activated dendrimers, and lipopolyplexes (Figure 2)2.
The researchers leverage a picodroplet-based microfluidic method capable of automating the complete CRISPR ribonucleoprotein transfection workflow, eliminating the need for multiple handling and selection steps, as in classic genome-editing workflows. The findings show that single cells can be encapsulated into picolitre-sized droplets and incubated for prolonged timeframes to undergo downstream editing processes such as gene knockout, knockin, gene insertion, and modified gene expression (Figure 1). In the current study, the edited gene exhibited expression of green fluorescent protein (GFP).
Using an integrated system, actuated through an intuitive user interface, researchers can analyse and selectively sort GFP-positive picodroplets at high throughputs (Figure 3) then dispense into 96-well microtitre plates for recovery and expansion (Figure 4). Overall, the results of the study demonstrate the successful application of a picodroplet-based integrated platform to perform a complete genome-editing workflow (Figure 5).
Conclusion
Genome editing presents a realm of therapeutic opportunities for the targeted treatment of cancer and other genetic disorders. Researchers can combine automated picodroplet-based technologies with the high efficiency of the CRISPR-Cas9 system to optimise research and development efforts. Using this method, a population of cells in picodroplets, with editing occurring inside the droplet, will be generated, screened at a single-cell level for desired edits, and dispensed in a significantly shorter time.
These advancements in picodroplet-microfluidic technologies demonstrate that a complete end-to-end workflow solution for genome editing and cell engineering is highly achievable. The ability to perform transfection, selection, sorting, and dispensing on a single automated device will make the genome-editing pipeline more streamlined. These capabilities will generate reproducible and reliable data through reduced workflow timelines and improve the scalability of genome-editing processes.
Figure 1: Results show that, depending on the cell line, cells can be tested, retrieved, and seeded for clone outgrowth after up to 72 hours in picodroplets.
Figure 2: Results confirm that picodroplet generation and stability are not affected by different transfection reagents across all experiments.
Figure 3: Diagram of an integrated genome-editing workflow for the accurate and sensitive detection, soring, and dispensing of successfully edited clones
Figure 4: Scatterplot of FRET signal generated from analysing and sorting GFP-positive picodroplets
Figure 5: Image of colonies generated from fluorescent microscopy analysis, illustrating that the majority were expressing GFP.About the author
Olivia Hughes is a life science writer and Digital Marketing Associate at Sphere Fluidics. Sphere Fluidics develops and manufactures single cell analysis and monoclonality assurance systems to enable leading-edge research and accelerate biotherapeutic discovery and development.
References
- Genome Editing Market – Global Forecast to 2025. MarketsandMarkets. https://www.marketsandmarkets.com/Market-Reports/genome-editing-engineering-market-231037000.html Accessed July 30, 2021.
- Shvets E, Lui X, Hughes O and Craig F. Cell Engineering in Picodroplets (2021). https://spherefluidics.com/resources/application-materials/ Accessed July 30, 2021.
- Josephides, D et al. Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance. SLAS TECHNOLOGY: Translating Life Sciences Innovation, 25(2): 177–189 (2020).
- Ahmadi F, Quach A, and Shih S. Is microfluidics the “assembly line” for CRISPR-Cas9 gene-editing? Biomicrofluidics, 14(6): 061301 (2020).
- Li X, Aghaamoo M, Liu S., Lee D and Lee A. Lipoplex‐Mediated Single‐Cell Transfection via Droplet Microfluidics. Small, 14(40): 1802055 (2018).
