Suitable for any cell type maintaining high viability

The development of the CRISPR system as a gene editing tool has very quickly revolutionized life sciences (1), as recognized by the 2020 awarded Nobel Prize in Chemistry. 

The system relies on a ribonucleoprotein (RNP) complex, the so-called CRISPR-complex, which includes 2 components: the Cas (CRISPR associated) protein, most often Cas9, that carries the enzymatic activity, and the guide RNA (gRNA). The gRNA drives the complex to the genomic locus of interest by sequence homology of the guide RNA with the genomic DNA and forming an RNA-DNA duplex. The Cas9 protein can then exert its endonuclease activity that will induce a double-strand break (DSB) in the targeted DNA. DNA repair pathways will consequently be activated. Among them, two have been particularly well described and used by researcher to ultimately obtain desired mutations.  The first one is by direct ligation of the break ends without the use of a template, the so-called non-homologous end joining repair (NHEJ), which usually is prone to errors and leads to insertions and deletions of base pairs, collectively called indels, potentially resulting in a Knock-out (KO) of the targeted gene. The second option is to use a repair template-based homology-directed repair (HDR) pathway to precisely edit the targeted region according to the respective template. This allows to perform Knock-in (KI – introduction of a transgene) or very precise single nucleotide replacement.

It is no surprise that this broadly applicable and versatile technology has experienced an enormous adoption in nearly all molecular life science disciplines ranging from fundamental and pharmaceutical research to applications in agriculture and gene and cell therapy(2). Still, however, many challenges remain, for both in vivo and in vitro applications, and there is plenty of room for improvement to fully tap the potential of this technology.  With FluidFM, biomechanical properties such as adhesion can be readily and efficiently measured, allowing to gain deeper understanding, to design novel experimental approaches, and to explore solutions to modify and optimize the desired properties.

For a nice visualization of HDR and NHEJ, please check the image in:
Barman, A., Deb, B. & Chakraborty, S. A glance at genome editing with CRISPR–Cas9 technology. Curr Genet 66, 447–462 (2020). https://doi.org/10.1007/s00294-019-01040-3

Basic research

The general applicability of CRISPR among different species has opened up a multitude of new approaches for biological studies(2). Genomic alterations, in coding and non-coding regions, can be artificially introduced and hence be studied. Endogenous proteins can be tagged without the need of a transgene(3). 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(4). Endogenous gene expressions and epigenetic states can be altered and many more possibilities have been enabled by CRISPR-based methods.

Gene and cell therapy

The vast new possibilities in basic research enabled by CRISPR-technology will eventually lead to novel approaches in medicine and has been already widely used in translational research projects. The simplicity of use of the CRISPR tool makes it very easy to target and correct any disease causing mutation and makes the CRISPR technology particularly promising for gene and cell therapy approaches. 

The translation of results from fundamental research into clinic has however been challenged by several obstacles that raise safety concerns, in particular the possibility of off-target effects which could lead to malignant transformations.  Several approaches have been developed to minimize the number of these off-target mutations, involving better engineering of new Cas proteins and gRNAs(5). Furthermore, in vivo delivery methods for CRISPR-reagents are not yet optimal with regard to local targeting and packaging, e. g. AAV, the most widely used viral carrier, is hardly able to load more than the sequence of a full Cas9 and a short gRNA. More extensive changes requiring longer repair templates are hence not deliverable with this approach. Another concern is host immune response to the bacterial Cas9, which could lead to unwanted side effects. 

Nevertheless, the concept of a therapeutic use of CRISPR is extremely promising and unsurprisingly, a huge number of clinical trials are already ongoing, mostly involving cell-based therapies where genome editing is performed outside the human body. Most recently however, first clinical studies aiming at gene editing within the human body have started for a liver and an eye disorder have started(6).

Bioprocessing

CHO cells are the work horse for protein production. They are generally easy to transfect with the transgene coding for the protein of interest. However, to optimize the production yield and to lower the effort for downstream processing, like protein purification. CRISPR gene editing is investigated to optimize the cell’s metabolism, aiming to increase the yield of production of the protein of interest while reducing the generation of metabolic byproducts. 

Furthermore, targeting post-translational modifications pathways in CHO cells is also investigated to improve the biology characteristics of the produced proteins. For example, glycosylation pathway can be targeted in the case of the production of vaccine immunogens(7).

Agricultural science and industry 

A fundamental advantage of CRISPR-technology is the species independent mechanism. Hence, the technology is also applicable in plants and therefore offers great potential in agriculture and plant biotechnology(8). Though plant breeding is well established for most domesticated plants, the process is tedious and time-consuming and usually requires several generations, especially for more complex phenotypes where the optimization of several genes is required. A main focus in this space is the improvement of crop by increasing its yield, conferring resistance to diseases or herbicides, and improving its quality (e. g. by reducing the gluten content).

CRISPR HDR efficiency

Precise editing of the genome is needed for example for protein tagging, correcting a disease-causing mutation or introducing a specific transgene at a defined location. In these examples, the success of the CRISPR-induced mutation relies on the homology-directed repair pathway (HDR), an intrinsically low efficiency process.

Many approaches have already been explored to increase this efficiency, for example covalent binding of the repair template to the Cas9-gRNA RNP complex or synchronizing the cell cycle of the targeted cells(9). Most of the current CRISPR gene editing projects however focus on small edits or insertions with small repair templates, as only these have a satisfactory HDR efficiency.  

Editing larger portions of the genome is of great interest and needed when editing a cell to produce antibodies, or in immuno-oncology projects involving the development of CAR-T cells(10). However, delivering large repair templates is a major challenge. Conventional transfection methods (lipofection, electroporation, viral transduction) usually fail to deliver large repair templates efficiently together with the CRISPR/Cas system components.

Overcoming these limitations has led researchers to investigate other transfection methods. Microinjection can easily deliver large repair templates but is restricted to large oocytes/zygotes. FluidFM nano-injection with force feedback control of the nanosyringe penetration overcomes this issue and can easily deliver large molecules(11, 12).

Hard-to-transfect cells

As for any genetic material, CRISPR systems can be quite cumbersome to deliver to cells that are notoriously known to be hard-to-transfect, such as primary neurons, pluripotent stem cells (ESCs, iPSCs…), suspension cells (T and B-cells) etc(13). Therefore, finding a solution that can efficiently, gently, and safely deliver genetic material – or any kind of molecules – is particularly important for researchers in the field of immunology, development biology, neurosciences and others. 

While lentiviral vectors often offer a nice solution in term of efficiency, it unfortunately also comes with some drawbacks. The most important are the random insertion of the viral sequence into the host genome and the persistence of Cas9 expression. This is a major concern in therapeutics where viral delivery approaches have been shown to be responsible of severe pathologies(14, 15). This is also problematic in fundamental research, where random insertion can lead to biological consequences that can be difficult to distinguish from the effects induced by the CRISPR itself.

An alternative to viral transduction is transfection by injection. Though limited to a few hundred up to thousands of cells, this approach easily delivers any material to any type of cells. Conventional microinjection is only suitable for large cells and can be difficult to handle, leading to a lot of cell death. FluidFM force-controlled nanoinjection can overcome these limitations.

CRISPR Multiplex Gene Editing

Targeting several loci in a multiplexing strategy approach is a growing trend in the field of life sciences. Multiplex gene editing is for example required when studying or treating multi-genic disease, or for genome writing projects such as de-extinction projects(16). 

Editing a single locus with CRISPR/Cas system can be rather straightforward when working with standard cell lines. Multiplex locus editing however faces two major challenges: the first is the toxicity brought by multiple double-strand breaks (DSBs) into the genome, which induces DNA damage response from the cell and can lead to apoptosis. The second is the efficient simultaneous delivery of multiple gRNAs together with the Cas protein, into the same cell. Engineered Cas proteins have been developed to be able to read and process CRISPR arrays that encode multiple guide RNAs from a single plasmid. However, creating these arrays is requires complex and time consuming upstream molecular cloning steps, which prevent easy access and limit the number of gRNAs that can be delivered. Today the maximum number of genes that can be targeted in a multiplex editing experiment is limited to a few dozens, for example 25 with the Cas12a(17) or 30 targeted loci with Cas9(18).

FluidFM nanoinjection approaches could solve the complex simultaneous delivery of multiple gRNAs. Several hundreds of different gRNAs can theoretically be delivered in a single injection into a specific cell. DSB induced toxicity however still limits the number of loci that can be targeted per injection when using Cas9. Combining this nanoinjection approach with the use of engineered Cas proteins which do not induce DSBs might therefore help to push multiplex editing beyond current limitations. In that regard, base editors(19) or dCas9 fused to an engineered reverse transcriptase enzyme for prime editing(20) are particularly promising. 

Read the article "Novel Approaches to Overcome CRISPR In Vitro Delivery Challenges" for a more detailed elaboration.

Read now

	Lipofection	Electroporation	Viral transduction	Microinjection	FluidFM injection
Hard-to-transfect cells	-	+	+	+	+++
Multiplex editing	-	+	+	+	+++
HDR - large knock-in	-	+	+	+	+++
Genetic screens	-	-	+++	-	-
Autologous gene / cell therapy	-	+++	++	-	-
Allogeneic cell therapy (off-the-shelf)	-	+	++	-	+++
Transfection of million of standard cells	+++	+++	+++	-	-
Generation of animal models	-	-	-	+++	-

- Not recommended; + Works but with drawbacks; ++ Works but not optimal in every situation; +++ Method of choice

When millions of edited cells are needed, batch transfection methods are more appropriate. For example, lentiviral vectors are the method of choice when performing CRISPR genetic screens, due to the very high efficiency of transfection. In gene/cell therapy, electroporation would be preferred as viral vector often come with random insertion into the genome, which could bring undesirable effect when used in a patient.

Injection methods are particularly appropriate when few modified cells (several hundreds) are needed. This is the case for example when creating CRISPR KO/KI monoclonal cell line, when working with rare cells or when multiplex editing is needed.

1. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

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

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

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

5. Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S. & Yang, S.-H. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol. Ther. Nucleic Acids 4, e264 (2015).

6. Hirakawa, M. P., Krishnakumar, R., Timlin, J. A., Carney, J. P. & Butler, K. S. Gene editing and CRISPR in the clinic: current and future perspectives. Biosci. Rep. 40, (2020).

7. Byrne, G. et al. CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation. PLoS Biol. 16, (2018).

8. Eş, I. et al. The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated challenges. Biotechnol. Adv. 37, 410–421 (2019).

9. Aird, E. J., Lovendahl, K. N., St Martin, A., Harris, R. S. & Gordon, W. R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun. Biol. 1, 54 (2018).

10. Eyquem J, Mansilla-Soto J, Giavridis T, van der Stegen SJC, Hamieh M, Cunanan KM, Odak A, Gönen M, Sadelain M. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543:113–117 (2017).

11. Guillaume-Gentil, O. et al. Force-controlled fluidic injection into single cell nuclei. Small Weinh. Bergstr. Ger. 9, 1904–1907 (2013).

12. Guillaume-Gentil, O. et al. Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 32, 381–388 (2014).

13. Riedl, S. A. B. et al. Non-Viral Transfection of Human T Lymphocytes. Processes 6, 188 (2018).

14. Gaspar, H. B. et al. Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency. Sci. Transl. Med. 3, 97ra79 (2011).

15. Woods, N.-B. et al. Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood 101, 1284–1289 (2003).

16. Thompson, D. B. et al. The Future of Multiplexed Eukaryotic Genome Engineering. ACS Chem. Biol. 13, 313–325 (2018).

17. Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D. & Platt, R. J. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat. Methods 16, 887–893 (2019).

18. Vad-Nielsen, J., Lin, L., Bolund, L., Nielsen, A. L. & Luo, Y. Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. Cell. Mol. Life Sci. CMLS 73, 4315–4325 (2016).

19. Jeong, Y. K., Song, B. & Bae, S. Current Status and Challenges of DNA Base Editing Tools. Mol. Ther. 28, 1938–1952 (2020).

20. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).