CRISPR: Tips and Tricks for Efficient Delivery into Primary Cells and Cell Lines – Technology Networks
Lipofection, electroporation, nucleofection, viral transduction: there are numerous approaches to genome editing, be it on a DNA, RNA or protein level. However, many current technologies are time-consuming, inefficient, can trigger off-target effects, or are unsuitable for cells that are especially resistant to transfection.1
One emerging powerful method used across research, biotechnology and medicine is CRISPR/Cas9. Compared to technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR promises a versatile and precise mechanism for genome editing. The technology is able to efficiently transfect relevant cell types and target multiple sites. It’s these qualities that researchers are harnessing to explore the potential of CRISPR for applications such as biological research, early stage drug discovery and development of novel cellular therapeutics such as immunotherapy.
Reaching hard-to-transfect cells with CRISPR/Cas9
Some cells — primary and pluripotent stem cells, for example — are harder to transfect than others due to properties including endocytic uptake mechanisms, division rate, metabolic activity and immune response. To transfect such cells efficiently, genome editing processes must consider characteristics such as cell type, selection, transfection method, and substrate choice into their design.
Discovered in E. coli in 1987, the CRISPR/Cas9 pathway forms part of a naturally occurring genome editing process found within the bacterial immune system, and has since been adapted for use in eukaryotic cell genomics. So-called “guide” RNA (gRNA) binds to complementary stretches of targeted DNA, and forms a complex with the Cas9 endonuclease to direct DNA cuts at specific sites. Unlike ZFNs and TALENs, the DNA binding domain for CRISPR/Cas9 is an RNA rather than a protein. As a result, the domain and nuclease are not fused, producing a process that facilitates the design of new guide RNAs for new targets quicker and easier, and allows specific multiplexed targeting (as one Cas9 nuclease may be combined with several gRNAs).
Directly target cell nuclei with nucleofection
A key element underpinning the success of CRISPR/Cas9 is the efficiency at which gRNA and Cas9 are delivered to the target cell. Alternative methods include lipofection, which passively introduces DNA into cells via synthetic lipid complexes, and viral vectors, which can be painstaking to design and only support DNA-based substrates. CRISPR/Cas9, however, can deliver modulating components directly to the nucleus of a target cell using electroporation-based Nucleofection™ Technology. This technology takes advantage of a unique combination of the Nucleofector™ device, a transfection solution and optimized protocols for delivering substrates such as DNA, RNA, and ribonucleoproteins (RNPs) directly into the nucleus of primary cells and cell lines. Such a technique results in high transfection efficiency rates together with high cell viability after transfection.
The Nucleofector™ Technology has proven successful and efficient at delivering CRISPR substrates directly to the nuclei of hard-to-transfect cells via pre-optimized electrical pulses. Direct transfer improves the chances of achieving high transfection efficiencies (including in non-dividing primary cells), prompts faster gene expression, requires less DNA per transfection, and allows multiple samples to be processed simultaneously.
Utilize RNA and ribonucleoproteins to minimize toxicity
Some genome editing processes bring unwanted results by affecting genes other than those targeted, or triggering toxicity in sensitive cells. One relatively new method to reduce toxicity risk uses RNPs that are built in vitro from Cas9 protein and gRNAs, as a CRISPR substrate. The use of RNPs offers greater control over gene editing, helps overcome the issue of mRNA instability improves efficiency by allowing genome modification to begin immediately after transfection. In turn, this could bring high gene knockout efficiencies.2
Toxicity risk can also be lowered by delivering Cas9 as capped and poly-adenylated mRNA which has a shorter half-life than plasmid DNA. Using this approach, Cas9 mRNA is transfected and translated within the cytoplasm of the target cell and combines with co-delivered “single guide RNA” (sgRNA) before entering the nucleus. Studies suggest that sgRNA could be chemically modified to further enhance gene editing efficiency and facilitate greater gene disruption in hard-to-transfect cells such as T, CD34+ hematopoietic stem and progenitor cells.3
Enrich gene-edited cells via labeling and co-editing
It takes several days to identify gene-edited cells via magnetic pull-down assay or deep sequencing. Newer methods offer fast and robust alternatives. In one study, researchers demonstrated that non-invasive fluorescent labeling works as a marker to enrich cells that have been successfully transfected, increasing the likelihood of a gene editing event.4 They fluorescently labeled their donor DNA with Alexa 647, and subjected transfected human embryonic kidney cells and primary myoblasts to fluorescence-activated cell sorting.
Another study used a co-selection strategy without markers.5 In addition to editing a target gene, researchers coded for a point mutation at the ATP1A1 locus. This mutation prevented gene-edited cells from being affected by an ATP1A1 inhibitor (ouabain), but allowed researchers to subsequently enrich modified cells with ouabain at another unlinked locus of interest.
A versatile and precise approach to genome editing
Genome editing has seen significant innovation and progress since the introduction of CRISPR/Cas9, but cell resistance to transfection remains a challenge. However, by quickly delivering components directly to target cell nuclei, CRISPR technology brings versatility, precision and efficiency to genome modification, while also minimizing off-target effects and toxicity, and enabling quick, non-invasive cell enrichment. Using CRISPR, researchers can optimize their approach to modifying the genes of physiologically relevant cell types — including primary, stem and other cells that are especially sensitive or hard to transfect.
1. Gupta R.M., Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest. 2014 Oct;124(10):4154-61. doi:10.1172/JCI72992.
2. Seki and Rutz. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J Exp Med, 2018.
3. Hendel et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol, 2016.
4. Lee et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife 2017;6:e25312 DOI: 10.7554/eLife.25312.
5. Agudelo et al. Marker-free coselection for CRISPR-driven genome editing in human cells. Nat Methods, 2017.
Published at Thu, 30 Jul 2020 00:08:31 +0000