Cytosurge’s FluidFM technology facilitates multiplex CRISPR editing and monoclonal cell line development in less than three weeks

In collaboration with University Children's Hospital, Tübingen, the genomic loci of several genes were simultaneously targeted by a multiplexed FluidFM nano-injection into single CHO cells, directly delivering gRNA/Cas9 RNP complexes into the nucleus.

Cytosurge, one of the leading global providers for precision single cell manipulation, has announced that its pioneering, patented FluidFM nano-probe enabled CRISPR multiplexing to generate monoclonal multiple Knock-Out cell lines in less than three weeks starting from the day of transfection until the clones have been characterized.

Pharmaceutical research and biologics manufacturing rely on genetically modified cell lines with genes that have been modified to induce the desired phenotype. In conventional cell line development pipelines, several candidates are evaluated within an iterative process to obtain stable monoclonal cell lines, a process that currently requires twelve to fourteen weeks. In comparison, by favoring a “bottom-up” approach, FluidFM technology can pick a single cell that it has modified and generate a clone out of it – in less than three weeks.

University Children's Hospital, Tübingen, and Cytosurge recently generated monoclonal multiple Knock-Out cell lines using FluidFM technology. Genomic loci of several genes were simultaneously targeted by a multiplexed FluidFM nano-injection into CHO cells, directly delivering gRNA/Cas9 RNP complexes into the nucleus. Using the same system, the successfully transfected cells were isolated for further expansion – more than 90% of the isolated cells developed into a stable monoclonal colony. Read the Application Note.

“Fourteen days after transfection, the clones were collected for analysis by Sanger sequencing. Overall, 50% of the clones showed mutations in targeted loci,” said Dr. Justin S Antony, University Children’s Hospital, Tübingen. “As this data demonstrates, the FluidFM bottom-up approach drastically reduces the processing time for monoclonal cell lines with multiple Knock-Out from months to less than three weeks. In turn, this technology allows the great opportunity to speed up the process for recombinant proteins and vaccine production, which might be advantageous to confront pandemics like COVID-19.”

The technological advances brought by Cytosurge’s FluidFM technology into the field of single-cell gene engineering have the potential to solve some of the most demanding challenges that scientists are currently facing when they need to rapidly and efficiently develop monoclonal cell lines. Current methods are adequate when applied to the most common cell lines and gene engineering strategies. However, they quickly reach their limits when dealing with uncommon, rare or fragile primary cell types that are also known to be hard-to-transfect, or when complex experimental design - for example, CRISPR multiplexing - is needed.

“These results show that multiple gRNAs can be simultaneously delivered into selected single cells by FluidFM nano-injection,” said Dr. Paul Monnier, Life Science Application Scientist, Cytosurge. “We believe that CRISPR multiplexing with the presented method can be scaled up to simultaneously co-inject several tens of gRNAs and that the Cytosurge FluidFM bottom-up approach will significantly accelerate development cycles.”

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