FluidFM® - A Stable Cell Transfection Method to support CRISPR-Cas9 Gene Editing Tool

This article describes how the FluidFM single cell transfection method can be employed effectively to support CRISPR Cas9 gene editing tool for in vitro disease modeling and drug discovery applications. With its direct intra-nuclear delivery capability, the FluidFM OMNIUM can perform high throughput transfection for a stable and custom cell line development.

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About the importance of a stable cell transfection combined with an efficient gene editing tool for precision gene editing
What makes CRISPR, an effective gene editing tool for precise genome engineering?
What makes the FluidFM, a high efficiency and throughput cell transfection method?

About the importance of a stable cell transfection combined with an efficient gene editing tool for precision gene editing

Gene or genome editing refers to the action of modifying an organism’s genetic code. This allows genetic material to be added, removed, or altered at specific locations in the genome. Throughout the years, several gene editing techniques have been developed, allowing different gene editing approaches such as gene knock-in (KI) and knock-out (KO) to be performed specifically and reliably on the genome. One of them - the CRISPR Cas9 gene editing technique or tool - started a revolution in the life science and biomedical research. With it, new innovative technologies and tools have arisen in the field of cell transfection methods to push further the limits of genome engineering. One of them, the FluidFM technology, offers a direct intra-nuclear delivery of soluble compounds - including CRISPR Cas9 complexes, for a high throughput and efficient cell transfection. This combination has allowed researchers to speed up their genome engineering experiments. Before deep diving into gene editing tools and technologies, a short introduction to the principles of the most prominent system of genome engineering CRISPR-Cas9 is required.

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What makes CRISPR, an effective gene editing tool for precise genome engineering?

A simple working principle

CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats" of genetic information that bacteria employ as part of their defense against viruses [1]. In practice, CRISPR genome editing involves a set of actions to accurately cut DNA and allow natural repair mechanisms to occur. The technology is based on a guide RNA and the Cas9 endonuclease; the guide RNA recognizes target sites and the Cas9 endonuclease cuts and triggers the repair mechanism.

Compared to previous gene editing methods, CRISPR enables a simplified, more versatile and direct genome editing. The ability to insert a gene at a specific locus is tremendously useful, notably to study gene function and to dissect disease mechanisms. Hence, it is with no surprise that this broadly applicable and versatile technology has experienced an enormous adoption in various research areas.

CRISPR has now outstripped other methods in terms of ease of use and editing efficiency. As an example, CRISPR with its very high specificity and efficiency, is recommended for research areas where multiple simultaneous edits are required within the same cell line or organism. Nonetheless, CRISPR is not the solution to solve all and every challenge remaining in gene editing because it still has some limitations.

CRISPR-Cas9 Gene editing tool - FluidFM - Cytosurge

Delivery and off-target limitations

In gene editing, the success of an experiment relies on several parameters. The success rate is depending on the choice of gene editing technique and cell transfection method. The limitations mentioned previously demonstrate the need to combine CRISPR-Cas9 gene editing tool with a reliable and gentle single-cell transfection method. The efficiency of a single-cell transfection experiment is determined by several factors among which, the complexity of the gene edits, the ability to reduce off-target editing and the insurance to keep the cell alive while performing the transfection method.

The two latter remain a challenge for CRISPR, notably for clinical application. To overcome those limitations, Cytosurge employed its proprietary patented technology - the FluidFM - to offer unprecedented genome editing capabilities with a unique in vitro solution to perform direct intra-nuclear delivery that keeps the cell alive. This method allows to increase the efficiency and applicability of CRISPR across a variety of cell types and research fields.

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What makes the FluidFM, a high efficiency and throughput cell transfection method?

The FluidFM® delivers your CRISPR Cas9 complexes directly to where they are needed: the nucleus.

Seven decades ago, the complex evolution of genome engineering started with the discovery of the DNA double helix. Throughout the years, researchers uncovered different tools and technologies to perfect gene editing. The FluidFM OMNIUM illustrates today's best single cell transfection method to perform high-efficiency precision genome engineering. The FluidFM technology unites the unique features of microfluidics and force microscopy by combining closed microscopic channels with force sensitive probes. These probes have apertures down to 300 nm and allow to dispense or aspirate femtoliter volumes.

FluidFM as a Genome editing tool by Cytosurge

The FluidFM technology provides:

Direct intra-nuclear injection: Reach an unprecedented precision delivery and a high CRISPR HDR efficiency due to a ultra-gentle transfection method based on nano-injection.
High cell viability: The gentle injection or extraction ensure your cell viability. Especially suitable for hard-to-transfect cells and rare cell types.
Complex edits: Easily introduce multiple gene edits in one go while maintaining your cells alive. Fasten your stable monoclonal cell lines generation!
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What the FluidFM® together with CRISPR gene editing tool, can be used for?

The general applicability of CRISPR among different species has opened a multitude of new approaches for biological research [4]. Researchers use gene editing tools in their work to investigate several problematics, illustrating the broad applicability of gene editing.

In the literature, three common uses can be identified:

• Gene editing to analyze the function of a gene. Genomic alterations, in coding and non-coding regions, can be artificially introduced and hence be studied.

• Detecting new drug targets via genetic screening. Genetic screens can be performed with large-scale genetic loss- or gain-of-function experimental approach designed to discover genes or genetic sequences that elicit a specific function or phenotype [5]

• Tracking or tagging of a cell based on a fluorescent protein added onto a selected gene. Endogenous proteins can be tagged without the need of a transgene [6].

Those three functions are supporting scientists in their laboratory for a broad range of life sciences and biological applications.

Applications areas

A gene editing tool can have an impact in many different disciplines from medicine to agriculture. This section will focus on the potential impact of CRISPR gene editing tools in Medicine. The possibility to alter the genome provides researchers and clinicians alternative methods to investigate and improve disease prevention and treatment.

Drug Discovery

The feasibility and accessibility of CRISPR technology fastens drug discovery processes, notably for screening new drugs.

Diagnostics

With its strong specificity, CRISPR gene editing tool can be used to identify infectious and non-infectious diseases.

Disease Modeling

Genetic diseases originate from “errors” in the genome. Consequently, CRISPR with its high specificity, efficiency, and ease of use, can be employed to treat diseases that are driven by genetic changes. Gene editing is employed to model diseases, both in vivo and in vitro.

How to choose the right single cell transfection method to perform gene editing for your project?

Over the years, our team has identified two main needs for gene editing in the research community:

The versatility, accuracy and ease of use of the FluidFM OMNIUM - that allows you to perform as many gene edition as you want (KO, KI) while maintaining your cells alive and well!
The precision, reliability and reproducibility of a CRISPR-Cas9 cell line development service: you focus on your science, we take care of the rest!

Conclusions - Take Away Messages

Genome Editing

The action of modifying an organism’s genetic code with various techniques, tools, and technologies.

CRISPR-Cas9

Currently, the most versatile gene editing method available on the market.

FluidFM

The only CRISPR gene editing supporting technology which provides direct single-cell intra-nuclear injection, extraction or manipulation with a high HDR efficiency

References

[1] Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." science 337.6096 (2012): 816-821.

[2] Li, Hongyi, et al. "Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects." Signal transduction and targeted therapy 5.1 (2020): 1-23.

[3] Gaj, Thomas, et al. "Genome-editing technologies: principles and applications." Cold Spring Harbor perspectives in biology 8.12 (2016): a023754.

[4] Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

[5] Yu, J. S. L. & Yusa, K. Genome-wide CRISPR-Cas9 screening in mammalian cells. Methods San Diego Calif 164–165, 29–35 (2019).

[6] Zeng, F. et al. A Simple and Efficient CRISPR Technique for Protein Tagging. Cells 9, (2020).