CellEDIT: A New Approach to Vector-Free CRISPR Editing Starting From Single Cells

Challenges in CRISPR Genome Engineering and CellEDIT - a Precise and Elegant Solution

This blog post was written by Cytosurge, cell line development service provider that is dedicated to fulfilling the unmet need for implementing complex genetic designs into gene-edited mammalian cell lines.

Since Jennifer Doudna and Emmanuelle Charpentier’s Nobel Prize-winning discovery of CRISPR technology, this gene-editing tool has revolutionized biology by enabling precise DNA edits. However, using CRISPR for genome editing presents challenges, such as efficiently delivering CRISPR components into the cell nucleus, avoiding random vector integration and ensuring the correct balance of CRISPR components (stoichiometry) to improve multiplex editing and minimize off-target effects.

Traditional methods, such as viral vectors, can lead to random integration, reducing accuracy and consistency in gene editing. CellEDIT, described in a recent webinar by Dr. Tobias Beyer, Chief Scientific Officer and Application Scientist at Cytosurge, addresses these issues by offering a novel approach.

By using a FluidFM® Nanosyringe, the smallest needle in the world, to inject CRISPR/Cas9 ribonucleoproteic (RNP) complexes directly into the nucleus of single cells, CellEDIT bypasses the need for vectors. This allows for the simultaneous delivery of all necessary components at optimal concentrations, ensuring precise edits with minimal cellular disruption, and thus also minimizing off-target effects (Fig. 1). In summary, CellEDIT paves the way for more reliable and consistent genome editing.

Watch CellEDIT in action in this video:

The CellEDIT Service offers a streamlined workflow designed to maximize efficiency and accuracy: after the RNP complex is injected into a single cell, the process continues with the expansion of the edited cell into a clonal population, followed by validation through advanced sequencing methods such as Sanger sequencing and sequence deconvolution. This approach ensures that the desired genetic modifications have been achieved with high precision.

During the webinar, Dr. Beyer also showed several case studies demonstrating the practical applications of CellEDIT’s technology:

1. Multiplex editing in CHO-K1 cells: Simultaneous targeting of three genes (BAX, DHFR and FUT8) resulted in an efficient process, with a high survival rate of the edited cells and successful knockout of the intended genes. Importantly, the mutations did not impair the functionality of the cells. Read the full peer-reviewed publication in Biotechnology Journal (Fig 2.).
2. Knockout in hard-to-transfect cell lines: Despite the challenges, CellEDIT successfully knocked out the HPRT1 gene in SK-MES1 cells – a sensitive cell type with a long growth cycle extensively used in cancer research and essentiality screens, achieving successful clonal expansion and high editing efficiency. Download the application note.
3. Targeted deletion of exons to mimic a knockout mouse model: This project involved a customized editing strategy that deleted specific exons using a double guide RNA (gRNA) approach. The ability to precisely replicate animal models in cell lines provides researchers with valuable tools for studying gene function and disease mechanisms.
4. Inactivation of a transcription factor binding site in a gene regulatory region: This was challenging due to overlapping genetic elements, including exons of a neighbouring gene and an antisense transcript. However, thanks to the CellEDIT team’s scientific expertise and precise editing, the target site was inactivated without affecting surrounding transcripts, meeting the customer’s deadline and providing the exact modification needed to confirm the researcher’s hypothesis. Read the customer testimonial.

Meet the CellEDIT Team! Check out the article ‘Behind CellEDIT Service: The Experts Crafting Top-Quality Genetically Engineered Cell Lines for Researchers Worldwide’.

Conclusion: A Bright Future for Genome Engineering

As genome engineering continues to evolve, technologies like CellEDIT are paving the way for more precise and reliable gene editing. Whether addressing challenges in hard-to-transfect cells, targeting specific genomic regions or conducting complex knockout studies, the CellEDIT Service is already assisting life sciences and biology researchers worldwide by delivering high-quality gene editing in cellular models in only 10 weeks, ultimately meeting the growing demand for reliable cell line manufacturing and providing researchers with the tools they need to advance their research.

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For those who missed the live webinar and are interested in a deeper dive into the CellEDIT Service and the case studies, you can watch it on demand here.

For more information about the CellEDIT Service workflow, please visit the CellEDIT page, follow the CellEDIT Service LinkedIn page and subscribe to the newsletter for news, sales and more.

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