CRISPR delivery methods

CRISPR delivery

CRISPR depends on successful delivery to cells

CRISPR genome editing allows certain changes to be made to the genome of any cell type, but this is dependent on getting the CRISPR components into the cells that need to be edited. The basic components for CRISPR include a Cas enzyme and a guide RNA (gRNA). The gRNA itself may consist of one or two molecules of RNA. The Cas enzyme must complex with the gRNA to form a ribonucleoprotein (RNP). Getting all these components into the cells at appropriate concentrations and with good timing is crucial.

Plasmids vs. RNPs for CRISPR

The Cas enzyme and guide RNAs can be delivered as RNP complexes or can be expressed using a vector such as a plasmid or virus. Delivering the Cas enzyme and the guide RNA as an RNP complex has many advantages over expression vectors and therefore is the suggested approach.

Delivering RNPs involves binding of the Cas enzyme to the gRNA on the bench—outside of the cell—and then bringing it into the cell as a complete RNP. If delivering an expression vector such as plasmid or virus instead of RNP, there is greater risk of off-target cuts and/or integration of plasmid or viral vector DNA (including the Cas gene itself) in the genome of the targeted cells. RNP delivery also allows much faster initiation of gene editing than vector delivery.

Techniques for CRISPR reagent delivery

There are several different methods for delivering CRISPR components into cells. Major methods are shown in Table 1 and discussed below.

Table 1. Major approaches to delivery of CRISPR reagents into cells.

Electroporation

General electroporation: Electroporation uses a short pulse of electrical current to increase cell permeability temporarily, allowing molecules to be delivered into the cell. IDT recommends electroporation for general CRISPR genome editing in cell culture. The electrical current allows an entire RNP complex, including both a Cas enzyme and a CRISPR guide RNA, to pass through a cell's plasma membrane into the cytoplasm.

CRISPR gene editing efficiency can be increased by adding electroporation enhancers to the RNP reagents. IDT offers two electroporation enhancers, one for Cas9 RNPs  and the other for Cas12a RNPs. These electroporation enhancers are single-stranded DNA molecules with no homology to human, mouse, or rat genomes, and are unlikely to be incorporated into the target genomes. These DNA molecules are thought to act as carrier molecules to improve the rate of RNP delivery into cells during electroporation. Using electroporation enhancers can reduce the amount of RNP complex required, which in turn may reduce off-target genome editing and improve cell survival. Both of the electroporation enhancers have been shown to enhance overall gene editing efficiency with the    Neon™ (Thermo Fisher Scientific) and Nucleofector® (Lonza) systems. These instruments produce optimized electrical currents to deliver either plasmid or RNP from outside the cells directly to the nucleus. This is especially useful for genome editing in primary cells or cells that are difficult to transfect. The electroporation enhancers work only for electroporation, not for microinjection or lipofection.

Electroporation for homology-directed repair (HDR): Electroporation of RNP is also the method recommended by IDT scientists for HDR, using a single-stranded oligodeoxynucleotide (ssODN) as the donor DNA. The RNP complex and the donor DNA are electroporated together in a single step.

Lipofection

In lipofection approaches, lipid molecules are used to surround the molecule to be delivered. The resulting complex, the liposome, can fuse with a cell’s plasma membrane and release its contents into the cell. If a lipofection protocol for delivering non-RNP molecules (e.g., plasmids) into cells in culture is already known to have high efficiency in a certain cell type, it may also work for delivery of RNP in these cells. We currently recommend Lipofectamine® RNAiMAX or CRISPRMAX™ reagents (Thermo Fisher Scientific) for RNP delivery.

Other RNA-specific reagents may work as well. However, in our research, most classical plasmid and small RNA delivery reagents perform poorly with RNPs.

Microinjection

Microinjection techniques are usually used for oocytes and embryos. In comparison to electroporation and lipofection, microinjection is labor-intensive, costly, and time-consuming, requiring skilled lab workers and special equipment. It is slow because each embryo needs to be injected individually. Injecting fertilized zebrafish oocytes has resulted in precise CRISPR genome editing [1].

Nanoparticles and viruses

Nanoparticles, small particles made of a variety of materials, hold promise for delivery of CRISPR components in adult mammals [2,3]. Nanoparticles can deliver many kinds of compounds or drugs to certain cells based on a variety of technological approaches that have been developed in this area. Gold nanoparticles have been successfully used to deliver RNPs consisting of gRNA and either Cas9 or Cas12a to the brains of mice [4]. Nanoparticles using other materials such as zinc ions for delivery of CRISPR components are also being explored [5].

Several viral vectors have also been used for delivery of CRISPR-Cas genes and guide RNA genes, but these can introduce unwanted and potentially dangerous consequences, such as integration of Cas genes in the mammalian cell genome, so these vectors should be used only with great caution if at all [6].

Delivery of CRISPR and HDR components to plants and other non-mammalian organisms

There are many published protocols available for delivery of large molecules such as DNA and proteins to a wide variety of cell types. For many organisms, including zebrafish embryos and C. elegans, IDT customers have shared delivery protocols to support the research community. IDT scientists, in collaboration with other researchers, have described RNP delivery in zebrafish [1]. In plants, all of the delivery methods described above may be successful, in addition to several plant-specific methods such as particle bombardment and Agrobacterium plasmid delivery [7]. As with mammalian cells, it would be prudent to avoid methods that could introduce Cas genes into the plant cell genomes.

Products for CRISPR genome editing

References

1. Hoshijima K, Jurynec MJ, Klatt Shaw D, et al. Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish. Dev Cell. 2019;51(5):645-657.e4.
2. Qiu M, Glass Z, Xu Q. Nonviral Nanoparticles for CRISPR-Based Genome Editing: Is It Just a Simple Adaption of What Have Been Developed for Nucleic Acid Delivery? Biomacromolecules. 2019;20(9):3333-3339. 
3. Chen F, Alphonse M, Liu Q. Strategies for nonviral nanoparticle-based delivery of CRISPR/Cas9 therapeutics. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2020;12(3):e1609.
4. Lee B, Lee K, Panda S, et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat Biomed Eng. 2018;2(7):497-507.
5. Yang X, Tang Q, Jiang Y, et al. Nanoscale ATP-Responsive Zeolitic Imidazole Framework-90 as a General Platform for Cytosolic Protein Delivery and Genome Editing. J Am Chem Soc. 2019;141(9):3782-3786.
6. Chakraborty S. Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics. [version 1; peer review: 1 approved with reservations]. 2019. F1000Research 8(1846).
7. Zhang Y, Malzahn AA, Sretenovic S, et al.  The emerging and uncultivated potential of CRISPR technology in plant science. Nat Plants. 2019;5(8):778-794.