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).
All CRISPR components are delivered simultaneously and at the right concentration directly into the nucleus, paving the way to large genomic editing.
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.
GFP expression in a neuron 24h after nano-injection of plasmids into a mouse primary neuron. Courtesy of Jinan University, Guangzhou, China.
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.
Up until now, the delivery of genome editing agents like CRISPR-Cas9 (lavender) using delivery vectors (pink), and allowing it to simultaneously edit several target sequences in the genome (blue) often causes cells to fail in their functions and die. The collaboration investigates whether nanoinjection of the reagents into distinct locations of the cell, including its cytoplasm and nucleus, and in defined amounts, would allow higher-order multiplexed edits to the genome.
Courtesy of Wyss Institute at Harvard University.