Optimising CRIPSR gene editing of hard-to-transfect cells: the advantages of single cell control

CRISPR has transformed gene editing, but still presents challenges in hard-to-transfect cells, such as pluripotent stem cells and primary cells.1 The key to obtaining successful transfection in these cells lies in innovative workflows. Here Georges Müller, CEO and cofounder of SEED Biosciences, shares his perspective on why focussing on editing a single cell, rather than bulk cells, is a pivotal strategy to optimise CRISPR delivery.

Delivery of ribonucleoprotein (RNP) into cells is an essential factor for successful CRISPR gene editing. However, this is difficult to guarantee using traditional CRISPR gene editing methods, especially in hard-to-transfect cells. The standard CRISPR technique involves gathering a group of cells and then electroporating them, using short high-voltage pulses to overcome the barrier of their cell membranes. This allows bulk transfection of ribonucleoprotein (RNP) into the cells and then hopefully, nuclear translocation.

However, the traditional process can lack precision. Controlling the amount of RNP entering each cell is challenging, leading to uncertainty about the ratio of RNP that reaches any specific cell. This method of transfection is not uniformly distributed and achieving a 100% equal delivery is not guaranteed. Importantly for the researcher, there is no assurance that the RNP, once inside the cells, will reliably enter their nuclei. In hard-to-transfect cells, it is unlikely that this method will offer high transfection efficiencies. Additionally, post-transfection, the cells must undergo sorting, isolation, and subsequent cultivation, which can be a labour-intensive process.

New workflows in CRISPR gene editing

An alternative, more precise, approach involves isolating individual cells prior to transfection. This method has been developed by Cytosurge, and SEED Biosciences represents a step in this workflow.2 In this method, individual cells are seeded at the centres of multiple plates, and then the RNP is gently injected directly into the nucleus of each cell. This method provides careful control over the amount of RNP entering each cell, ensuring accurate measurement. The direct injection of RNP into the nucleus targets the specific site where it is needed, rather than broadly injecting it into the cell. Therefore, this meticulous method significantly improves the chances of achieving high transfection efficiencies in hard-to-transfect cells.

In addition, by adopting a gentle and precise approach, injecting directly into the nucleus means the utilisation of up to approximately 100 times less RNP compared to traditional methods. This not only benefits the project budget but also supports the quality of the cell editing process.

Once transfection has occurred, the cells can be grown to produce large monoclonal cell lines. Starting from a single cell rather than bulk cells eliminates the need for post-transfection cell sorting, saving hands-on time and ensuring 100 percent monoclonality. This holds particular significance, as it is a prerequisite for FDA approval in biotechnology that any cell clones must originate from a single-cell progenitor.

Read more here on Drug Target Review: https://www.drugtargetreview.com/article/147509/optimising-crispr-gene-editing-of-hard-to-transfect-cells/


[1] https://www.genengnews.com/insights/strategies-for-optimizing-crispr-cas9-delivery-into-hard-to-transfect-cells/#:~:text=Overcoming%20the%20challenges%20involved%20in,selection%20for%20successfully%20edited%20cells.

[2] Rosen, H. (2023) ‘The Ins and Outs of Single Cell Gene Editing’, New Matter. [Podcast]. Available at: https://slas.buzzsprout.com/951535/12714008-the-ins-and-outs-of-single-cell-gene-editing-sponsored-by-molecular-devices (Accessed 24th January 2024).