A. Kerjouan, S. Mailand, A. Migenda, K. Keevend, T. Ypma, S. Connolly, L. Rizzo, R. Lafleur, M. Milla, D. Ossola, T.A. Beyer
Cytosurge,
Switzerland
Keywords: FluidFM, live-seq, sc-RNA sequencing, genome engineering, mechanobiology
Summary:
FluidFM® is an emerging technology that integrates microfluidics with force feedback control allowing gentle manipulations of single cells. The technology consists of a force sensitive hollow cantilever with a sharp pyramidal tip (Nanosyringe) or a cup shaped opening (Micropipette) combined with a microfluidics pressure control unit. The Nanosyringe can either inject or withdraw liquids into/from cells and the Micropipette can be used to pick and relocate single cells by applying negative pressure. In the context of genome engineering, FluidFM® Nanosyringes are used to deliver CRISPR/Cas ribonucleoprotein (RNP) complexes directly into the nucleus of single cells. This approach, coined CellEDIT, provides unprecedented control on the amount and stoichiometry of RNP delivered to individual cells enabling multiplex editing and increases precision. By delivering the RNP directly to the nucleus of single cells, our approach eliminates cell toxicity caused by chemical and physical transfection agents as well as the use of viral vectors. Here, we present proof of concept studies, where we simultaneously targeted three individual genes in CHO-K1 cells or successfully edited a single locus in hard-to-transfect cell lines. Currently, we are expanding the CellEDIT workflow to co-deliver DNA repair templates to generate point mutations and insertions of large transgenes into distinct genomic loci. We envision that our workflow will enable us to move from genetically engineered to truly genetically designed cell lines. Further, cytoplasmic biopsies from cultured single cells can be obtained without impairing the cell’s viability using FluidFM® Nanosyringes. From these cytoplasmic biopsies, transcriptomes can be generated using low input library generation protocols. This novel approach coined Live-seq enables to obtain truly temporally resolved transcriptomes or to directly link cellular phenotypes to the gene expression pattern of individual cells. Currently, we are extending the cell repertoire, streamlining the process and optimize the library generation protocol to facilitate the application of Live-seq. In summary, we present the FluidFM® OMNIUM, a semiautomated platform that enables gentle manipulation of single cells to achieve CRISPR mediated genome designing at the single cell level while simultaneously recording the effects of the genome alteration at the transcriptome level.