Single Cell Transfection Method with CRISPR Cas9 Gene Editing Tool
Explore a unique and vector-free single cell transfection method suited for 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 Technology can perform high throughput transfection for a stable and custom cell line development.
Discover:
How important is a stable cell transfection combined with an efficient gene editing tool for gene editing?
![Gene Editing Tool FluidFM by Cytosurge Gene Editing Tool FluidFM by Cytosurge](https://cdn.cytosurge.com/web/image/348098/Cell%20Transfection%20Methods_Cytosurge-FluidFM_8.jpg)
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.
![CRISPR-Cas9 Gene editing tool - FluidFM - Cytosurge CRISPR-Cas9 Gene editing tool - FluidFM - Cytosurge](https://cdn.cytosurge.com/web/image/347501/Gene-Genome-Editing-Tool_FluidFM_Cytosurge%20%287%29.jpg)
Delivery & Off-Target Limitations
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.
What makes the CellEDIT workflow, a unique single-cell transfection method?
The FluidFM technology 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 technology illustrates today's best vector-free 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 FluidFM as a Genome editing tool by Cytosurge](https://cdn.cytosurge.com/web/image/328577/FluidFM-direct-intra-nuclear-injection-600x386-min.jpg)
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!
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.
Resources
Technical
Media & Downloads
On-Demand CellEDIT's CRISPR Cell Line Engineering Webinar
CellEDIT's Application Note n*1 - Introducing the CellEDIT Workflow
CellEDIT's Application Note n*2 - Overcoming the hard-to-transfect cell line hurdle
Open Access Publication featuring CellEDIT: Antony, Justin S., Anabel Migenda Herranz, Tahereh Mohammadian Gol, Susanne Mailand, Paul Monnier, Jennifer Rottenberger, Alicia Roig‐Merino et al. " Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9" Biotechnology Journal 19, no. 4 (2024): 2300505.
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).