With traditional delivery methods, such as lipofection or electroporation, the success of CRISPR-Cas mediated gene editing is often subpar due to poor cell viability and low transfection efficiency rates. Furthermore, precision editing by homology directed repair (HDR), occurs at even lower ratios. This is especially critical when working with rare or hard-to-transfect cells such as iPSCs, neurons or cardiomyocytes, making genome editing tedious.
At Cytosurge, we have developed FluidFM technology to improve CRISPR delivery by gently delivering all the required components directly to the nucleus of the cells. Thus, we provide scientists with a state-of-the-art tool allowing them to significantly enhance gene editing rate, optimize on/off-target effects and even enable easy multiplexing of CRISPR mutations.
Direct intra-nuclear delivery with FluidFM technology.
FluidFM technology unites microfluidics and force microscopy by introducing microscopic channels into force-sensitive probes. This unique combination enables the handling of liquid volumes at the femtoliter scale, as well as force-controlled manipulations of microscopic objects.
Thanks to the microfluidic channel inside FluidFM probes, soluble molecules can be dispensed through a sub-micrometer aperture at the tip. At the same time, the sensitive force feedback system provides a reliable distinction between gentle contact with cell membranes and their perforation.
FluidFM for CRISPR delivery
Multiple parameters can influence the efficiency of CRISPR gene editing. Amongst them, precise temporal and spatial control of the localization of the Cas9/gRNA ribonuclear protein (RNP) complex and the repair template is essential.
Generally, the delivery of the RNP complexes and the HDR template into the nucleus represents a major obstacle in the traditional gene editing workflow. Current methods deliver material to the cytoplasm first, as it passes through the cell membrane. However, to efficiently edit the genome, the materials must be transported to the nucleus of the cells, affecting the stability of the HDR template and the RNP. With FluidFM technology, we can elegantly bypass the cytoplasmic and nuclear membranes and directly deliver, through highly automated nano-injection, the material to the nucleus. This ensures that the RNPs and repair template reach their targets. The specific probe used for the direct delivery of CRISPR-Cas complexes into a cell is our FluidFM nanosyringe. It allows injection into single nuclei without compromising cell viability. The very sharp apex and the around 600 nm aperture at the front side of the pyramidal tip guarantees gentle perforation and injection of compounds with a wide range of molecular weights and densities.
In more sophisticated projects, researchers aim to edit several loci in the same cell simultaneously. Whether it is only a handful of genes to be targeted – for example to optimize new antibody production - or several hundred - for example in genome writing -, it is a very tedious process to successfully obtain mutations of all the targets in the same cell.
Using a FluidFM nanosyringe, we can deliver as many different CRISPR-CAS9 RNP complexes as desired, therefore allowing easy and instantaneous introduction of tens to thousands of different gRNA in the same single cell.
One of the main concerns of CRISPR gene editing is the possibility of off-target effects. Being a stochastic process, off-target effects increase upon exposure time and Cas9 concentrations (Hsu et al. Nature Biotech, 2013, Fu et al., Nature Biotech 2013, Lin et al., eLIFE 2014).
It is yet unknown how many RNP complexes are required for a successful integration of the desired change in a cell, whilst needing to minimize the numbers of off-target mutations. This is due to the fact that with conventional methods such as electroporation it is not possible to calculate how many molecules penetrate the cells and reach the nucleus.
With FluidFM technology, we can precisely determine the injected volume per cell, at a femtoliter scale. This allows us to precisely link the efficiency and number of off-target mutations to the effective number of RNP complexes delivered to the nucleus.
FluidFM technology has been successfully used in a wide range of applications, including cell adhesion measurement, cell isolation, nano-injection into cells or cell extraction. We foresee major impact of this technology, not only in the field of gene editing, but also in drug development or neurosciences.
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