Viral Generation, Packaging, and Transduction on a Digital Microfluidic Platform.

Viral-based systems are a popular delivery method for introducing exogenous genetic material into mammalian cells. Unfortunately, the preparation of lentiviruses containing the machinery to edit the cells is labor-intensive, with steps requiring optimization and sensitive handling. To mitigate these challenges, we introduce the first microfluidic method that integrates lentiviral generation, packaging, and transduction. The new method allows the production of viral titers between 106 and 107 (similar to macroscale production) and high transduction efficiency for hard-to-transfect cell lines. We extend the technique for gene editing applications and show how this technique can be used to knock out and knock down estrogen receptor gene─a gene prominently responsible for 70% of breast cancer cases. This new technique is automated with multiplexing capabilities, which have the potential to standardize the methods for viral-based genome engineering.

Is microfluidics the "assembly line" for CRISPR-Cas9 gene-editing?

Acclaimed as one of the biggest scientific breakthroughs, the technology of CRISPR has brought significant improvement in the biotechnological spectrum-from editing genetic defects in diseases for gene therapy to modifying organisms for the production of biofuels. Since its inception, the CRISPR-Cas9 system has become easier and more versatile to use. Many variants have been found, giving the CRISPR toolkit a great range that includes the activation and repression of genes aside from the previously known knockout and knockin of genes. Here, in this Perspective, we describe efforts on automating the gene-editing workflow, with particular emphasis given on the use of microfluidic technology. We discuss how automation can address the limitations of gene-editing and how the marriage between microfluidics and gene-editing will expand the application space of CRISPR.

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation.

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in-house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knockout transfections.

An automated microfluidic gene-editing platform for deciphering cancer genes.

Gene-editing techniques such as RNA-guided endonuclease systems are becoming increasingly popular for phenotypic screening. Such screens are normally conducted in arrayed or pooled formats. There has been considerable interest in recent years to find new technological methods for conducting these gene-editing assays. We report here the first digital microfluidic method that can automate arrayed gene-editing in mammalian cells. Specifically, this method was useful in culturing lung cancer cells for up to six days, as well as implementing automated gene transfection and knockout procedures. In addition, a standardized imaging pipeline to analyse fluorescently labelled cells was also designed and implemented during these procedures. A gene editing assay for interrogating the MAPK/ERK pathway was performed to show the utility of our platform and to determine the effects of knocking out the RAF1 gene in lung cancer cells. In addition to gene knockout, we also treated the cells with an inhibitor, Sorafenib Tosylate, to determine the effects of enzymatic inhibition. The combination of enzymatic inhibition and guide targeting on device resulted in lower drug concentrations for achieving half-inhibitory effects (IC50) compared to cells treated only with the inhibitor, confirming that lung cancer cells are being successfully edited on the device. We propose that this system will be useful for other types of gene-editing assays and applications related to personalized medicine.