CRISPR cell line development Use Cases with FluidFM®
Accelerate CRISPR gene editing and cell line development.
Transient expression of target proteins with mRNA
Human Dermal Fibroblast injected with GFP mRNA. 70% of injected cells expressed GFP 24h after injection.
Transient mRNA transfection helps to give fundamental insights on protein functions at a single cell level, such as protein expression and intercellular communication.
mRNA transfection using FluidFM nano-injection results in:
Higher transfection efficiency
Lower cell toxicity
Human dermal fibroblast expressing GFP, 24h after injection.
Generation of monoclonal multiple KO clones in <3 weeks
Start from a single cell clone and get your monoclonal cell line within 3 weeks, compared to 10-14 weeks with conventional approaches.
Injection of multiple gRNAs & Cas9
Multiple gRNAs are simultaneously delivered with the Cas9 into CHO cells. A fluorescent marker is co-injected to verify successful delivery.
Hands-on time: 30 minutes - from the loading of the FluidFM Nanosyringe until the injection of 50 cells is completed
Isolation of the clone candidate
Selected cells are isolated into separate wells. Visual proof of single cell deposition ensures monoclonality.
Hands-on time: 30 minutes - for the preparation of the FluidFM Micropipette and the isolation of 12 injected cells expressing the fluorescent marker
As of day 3:
Clone expansion and analysis
Of the isolated clones, 90% developed into monoclonal colonies and 50% thereof showed mutations in targeted loci.
Strong reduction in complexity and development time of genetically modified cell lines starting from few nano-injected cells
Direct intra-cellular delivery & volume measurement
Co-delivery of two plasmids
pEGFP-UHRF1 and pmCherry-TRIM21 (2 plasmids) co-injected into adherent CHO-K1 cells. The nuclear expression of the human UHRF1 protein is observed (3h post injection), as well as the cytoplasmic expression of the human TRIM21 protein.
Intra-nuclear CRISPR delivery
Mouse primary hepatocyte injected with CRISPR-Cas9 RNP complexes.
Measured volumes of fluorescent CRISPR-Cas9 complexes after injection into mouse primary hepatocytes.
Successful transfection of primary neurons with plasmids
With our system, even primary neurons can be transfected successfully and highly efficiently. The FluidFM nanosyringe directly delivers the complexes into primary neurons - gently and autonomously.
Conventional delivery methods applied to neurons are often inefficient, expensive and toxic to the neuron.
GFP expression in neurons 24h after nano-injection of plasmids in a mouse primary neuron. Courtesy of Jinan University Guangzhou, China.