CRISPR: Novel Methods to Combat In Vitro Delivery Challenges

The development and discovery of the CRISPR system as a gene editing technique has been a key innovation in life science, from agricultural sciences, human health in gene and cell therapy to fundamental biology, where it is now employed as a common technology (1).

Nevertheless, many challenges in the safety and delivery of CRISPR are yet to be solved to fulfill its potential for both in vitro and in vivo applications.

In the field of gene therapy, safety is a major concern. Off-target mutations of the genome must be reduced (2). This also holds true in the field of fundamental science. For example, researchers must ensure that the observed phenotype is produced by the targeted mutation rather than by off-target sites when investigating a signaling pathway.

Delivering the CRISPR/Cas system into the nucleus of the targeted cells is another key challenge. In vivo, it is required to travel through multiple barriers in order to target a particular cell within a living organism. In vitro, low efficiency techniques and cytotoxicity often restrict delivery of CRISPR/Cas system components into cell nuclei. Batch transfection utilizing electroporation or lipofectamine is ideal when performing single knock out in immortalized cell lines, which requires a single guide RNA (gRNA) along with the Cas9 protein. The intrinsic cytotoxicity of these widespread techniques is overcome by the high quantity of cells that are available. In turn, these techniques are much less suitable when dealing with large repair templates for homologous directed repair (HDR), with cells that are difficult to transfect, or for multiplexing projects where multiple gRNAs are needed. Here, the use of alternative, cutting-edge transfection techniques, like direct nuclear nano-injection, offer a suitable solution.

Challenge 1: Delivering CRISPR/Cas systems into hard-to-transfect cells

Traditional delivery techniques like electroporation or lipofection can be:

  • Highly toxic
  • Very ineffective

This is true for the majority of primary cells, for example, stem cells and neurons.

Viral transduction can solve these two main challenges, particularly when employing lentiviruses. The relatively low toxicity and high efficiency of viral transduction have positioned it as a preferred method, for example, when millions of edited cells are required when working on large scale CRISPR genetic screening projects (3). Viral transduction has its own limitations, which make it inappropriate for a number of applications, particularly in therapeutics. The most crucial are undoubtedly the persistence of Cas9 expression and the random insertion of the viral sequence into the host genome. This is a main concern in therapeutics, where the safety of viral delivery techniques is a major issue and has been proven to cause severe pathologies (4-5). In fundamental research, random insertions are also problematic as they might cause phenotypes that can be challenging to distinguish from the effects caused by the CRISPR itself. An additional limitation of this approach is that viral particles are a biohazard and require highly specialized instrumentation and biosafety laboratories (6).

Researchers in hematology, immunology, biologics production and CAR T cell therapy projects frequently work with nonadherent cells. Some of these cells, for example, T or B cells, are known to be very challenging to transfect (7). For researchers in these fields, it is especially important to discover a tool that can gently, safely, and efficiently deliver a genetic material or any kind of molecule into such cells.

For such hard-to-transfect cells, the use of novel injection techniques could be one solution. Microinjection protocols are well established for zygotes and oocytes. Due to limited automation potential and low viability, however, this technique cannot efficiently and safely deliver into other mammalian cells.

In this case, novel technologies such as force-feedback-controlled nano-injection could solve the limitations in both automation and viability, thus providing a transfection solution for primary cells like embryonic stem cells, neurons and T cells (8-9).

Cell line development with hard-to-transfect cells

When producing a genome-engineered cell line, traditional top-down techniques transfect millions of cells and then go through a time-consuming and iterative selection procedure until they can locate, isolate, and expand the clone of interest.

This method is ideal when working with immortalized cell lines, but researchers working with precious or rare primary cells often cannot afford to lose thousands of cells in this top-down approach.

Conversely, bottom-up methods begin with few cells where each is monitored to isolate and expand only the selected cells of interest that have been transfected.  Hence, high efficiency in the transfection process and isolation of specific cells are crucial. In this regard, new technologies that carry out automated cell isolation and nano-injection are highly promising (8, 10).

Challenge 2: Delivering large repair templates for HDR

In applications like protein tagging, when accurate editing of the genome is required, the efficacy of the CRISPR-induced mutation depends on the homologous-directed repair (HDR) pathway, a process that inherently lacks efficiency.

Numerous techniques have already been investigated to enhance this efficiency. For example, synchronizing the cell cycle of the targeted cells or covalent binding of the repair template to the Cas9-gRNA RNP complex are two of these approaches (11).

However, the majority of contemporary CRISPR gene editing projects concentrate on insertions with small repair templates or small edits, as only these have a sufficient HDR efficiency.

Delivering large repair templates still is a key challenge, despite the fact that there is significant interest and potential in editing larger areas of the genome. For example, large insertions are required when editing a cell to generate antibodies or in immune-oncology projects, which include CAR T cell development. Techniques like electroporation or lipofection normally fail to efficiently deliver large repair templates, along with the components of the CRISPR/Cas system, while viral particles are also limited in their packaging size and pose safety risks when used as a transfection tool.

The use of injection as a transfection technique has been investigated by scientists who aim to achieve large edits. Microinjection is highly effective for the delivery of large repair templates but is limited to zygotes or oocytes as this technique is challenging to use with other mammalian cells.

FluidFM, a technology that allows automated nano-injection with force feedback control of the nanosyringe, solves this challenge and can efficiently deliver large molecules (9).

Figure 1. Delivering CRISPR/Cas systems into the nuclei of targeted cells. Newly developed methods, such as quantifiable force-controlled nano-injection, can help to overcome this challenge when working with hard-to-transfect cells, such as mouse primary hepatocytes, with large repair templates or in multiplex editing projects. Image Credit: Cytosurge AG

Challenge 3: CRISPR multiplexing

Targeting multiple loci in a multiplexing strategy technique is an emerging trend in the field of life science, whether for the transcriptional regulation of multiple targets or for multi-locus editing. This is especially important for multi-genic disease research or genome writing projects, for example, de-extinction projects (12).

Although editing a single locus with CRISPR/Cas system is routinely done, editing multiple loci in the same cell is by far more challenging. Two key challenges are posed when multiplexing. The first is the toxicity caused by several double strand breaks (DSBs) in the genome, which elicits a DNA damage response by the cell that eventually leads to apoptosis, i.e., the death of the cell.

The second is delivering several gRNAs along with the Cas protein at the same time into the same cell. Engineered Cas proteins have been created with the ability to process and read CRISPR arrays that encode several guide RNAs. The number of gRNA is however limited in these arrays as their creation relies on challenging upstream molecular cloning procedures.  

At present, the maximum quantity of genes that can be targeted in a multiplex editing investigation is restricted to a few dozen. For example, 30 targeted loci with Cas9 or 25 with the Cas12a (13-14).

Nano-injection techniques like FluidFM have the potential to deliver hundreds of unique gRNAs through a single injection into a particular cell (8) and are thus able to provide a solution in the delivery of multiplex gRNAs. Toxicity may still restrict the number of loci that can be edited for each treatment when utilizing Cas9. Other gene editing approaches with engineered Cas proteins which do not involve DSBs may help to solve this toxicity limitation in multiplex editing. In that respect, dead Cas9 fused to an engineered reverse transcriptase enzyme for prime editing or base editors are highly encouraging (15-16).

Challenge 4: Minimizing off-target mutations

Off-target mutations arise from the Cas proteins cutting the genome at non-targeted loci (2). This poses a concern with safety risks when employing the CRISPR/Cas system for therapeutics. Multiple techniques have been designed to reduce the prevalence of these off-target mutations, including optimized engineering of novel gRNAs and Cas proteins (17).

CRISPR off-target mutations are understood to be a stochastic process that increases upon Cas9 availability and exposition time (18). Conventional delivery techniques like lipofection or electroporation do not provide accurate control over the number of molecules that are delivered into the nucleus.

New delivery tools, for example, quantifiable nano injection techniques like FluidFM, are highly promising as they would enable a precise dosage to be defined to reduce off-target mutations while enhancing CRISPR efficiency (8).

Looking ahead

It is critical to optimize the ability to deliver measurable quantities of the CRISPR/Cas system components into the nuclei of cells, such as B cells, embryonic stem cells, or neurons, in a gentle procedure that upholds high cell viability.

It will open new horizons for applications in fundamental research and therapeutics development. It could also facilitate some of the more exotic investigations, for example, de-extinction projects, where high multiplexing capabilities are required.

Novel technologies solving these delivery challenges, such as newly engineered Cas proteins and automated force-controlled nano-injection, will help to optimize CRISPR efficiency and will open new applications in the field of gene editing.

References and further reading

  1. Jinek M et al, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science 337(6096): pp816-21, 2012
  2. Zhang X-H et al, Off-target effects in CRISPR/Cas9-mediated genome engineering, Mol Ther Nucleic Acids 4(11): e264, 2015
  3. Yu JSL and Yusa K, Genome-wide CRISPR-Cas9 screening in mammalian cells, Methods 164-165: pp29-35, 2019
  4. Gaspar HB et al, Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency, Sci Transl Med 3(97): 97ra79, 2011
  5. 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(4): pp1,284-9, 2003
  6. Schlimgen R et al, Risks associated with lentiviral vector exposures and prevention strategies, J Occup Environ Med 58(12): pp1,159-66, 2016
  7. Riedl SAB et al, Non-Viral Transfection of Human T Lymphocytes, Processes 6(10): p188, 2018
  8. Guillaume-Gentil O et al, Force-controlled fluidic injection into single cell nuclei, Small 9(11): pp1,904-7, 2013
  9. Guillaume-Gentil O et al, Force-controlled manipulation of single cells: from AFM to FluidFM, Trends Biotechnol 32(7): pp381-8, 2014
  10. Guillaume-Gentil O et al, Isolation of single mammalian cells from adherent cultures by fluidic force microscopy, Lab Chip 14(2): pp402-14, 2014
  11. Aird EJ et al, Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template, Commun Biol 1(1): pp1-6, 2018
  12. Thompson DB et al, The future of multiplexed eukaryotic genome engineering, ACS Chem Biol 13(2): pp313-25, 2018
  13. Campa CC et al, Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts, Nat Methods 16(9): pp887-93, 2019
  14. Vad-Nielsen J et al, Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes, Cell Mol Life Sci 73(22): pp4,315-25, 2016
  15. Jeong YK et al, Current Status and Challenges of DNA Base Editing Tools, Mol Ther: 2020
  16. Anzalone AV et al, Search-and-replace genome editing without double-strand breaks or donor DNA, Nature


Produced from materials originally authored by Dr. Paul Monnier from Cytosurge.

Cytosurge AGAbout Cytosurge AG

Cytosurge AG develops, manufactures, and distributes state-of-the-art nanotechnology solutions and systems based on its patented FluidFM® technology. At the heart of the technology are the hollow FluidFM probes, which have apertures down to 300 nm, enabling the handling of femtoliter volumes.

Cytosurge brings with its FluidFM solutions significant benefits to a wide range of applications in life sciences, biophysics and mechanobiology. Unique benefits include quantitative volume measurements of injected compounds into single cells during drug development, improved CRISPR gene editing by direct delivery into the nucleus, isolation of selected cells directly from confluent culture, 2.5D nano-printing down to submicron levels, or single-cell adhesion and colloidal probe measurements.

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Last updated: Jan 26, 2021 at 8:22 AM


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