CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation
June 20th, 2019
Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.
Safely and effectively delivering Cas9 elements into cells is critical for gene editing based therapies. The protocols we show here display a novel approach in that direction. The protocol utilizes a new electroporation method called tube electroporation.
This leads to high cellular survival rates as well as gene editing rates. The tube electroporation method is virus, drug, and the reporter enrichment free. These are preferred in clinical applications, so with gene editing based therapies.
The method is universally applicable to many cell types. For example human hematopoietic cells, primary T cells, and other cells can all be edited using our method. This tube electroporation method uses a thin tube.
If you're a first time user, you can try GFP expressing plasmid DNA. Demonstrating the procedure will be Linyuan Ma, a postdoc from my laboratory. To begin this procedure acquire human iPSCs from the American Type Culture Collection.
Culture the iPSCs on artificial extracellular matrix with feeder-free cell culture medium in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide following the supplier's instructions. Two hours prior to transfection treat the iPSCs with 10 micromolar Rho-associated coiled-coil containing protein kinase inhibitor Y-27632. When transfecting, dissociate iPSCs with cell detachment solution to single cells at 37 degrees Celsius for five minutes.
Then count the number of cells. To establish a rabbit fibroblast cell culture obtain an ear skin biopsy from the tip of a rabbit ear. Rinse the tissue twice with DPBS containing 5%penicillin-streptomycin.
Transfer the rinsed ear tissue to a new six centimeter tissue culture dish and cut the tissue into small pieces, each about one by one millimeter. Add a few drops of FBS to prevent the tissue from drying out. After this, spread the shredded tissue in a 10 centimeter tissue culture dish and add 10 milliliters of culture medium.
Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for three to five days. Three to five days after plating use Trypsin-EDTA to digest the cells at 37 degrees Celsius for two minutes, then count the number of cells. After designing and synthesizing the gRNAs and donor oligos, prepare the cells as previously described.
Resuspend 20 to 30, 000 cells in 20 microliters of electroporation buffer. Pipette up and down carefully to produce a single cell suspension. For Cas9 RNP transfection, pre-mix two micrograms of Cas9 NLS protein with 0.67 micrograms of gRNA at room temperature for 10 to 15 minutes.
Gently mix the formed RNP complex with two micrograms of ssODN with cells. Using universal pipette tips from the electroporation kit, transfer the cell mixture to a 20 microliter electroporation tube. Place the electroporation tube into the slot of the electroporator and press go to finish.
A successful electroporation cycle is indicated by the pulse report on the display screen of the electroporator. After the electroporation transfer the human iPS cells to one milliliter of pre-warmed Y-27632 containing culture medium as previously described. Then plate the resuspended cells in one well of a 12-well cell culture plate.
Continue culturing the plate, making sure to change the culture medium every day. Y-27632 is removed from the human iPSC culture medium 24 hours post-electroporation. 72 hours after electroporation, harvest the cells.
For a rabbit fibroblast cells, use Trypsin-EDTA to digest the cells from the culture plate. For human iPSCs use cell detachment solution to digest the cells from the culture plates. Briefly centrifuge the samples at 1000 rpm for five minutes, and resuspend the cells with 350 milliliters of lysis buffer.
Incubate overnight at 55 degrees Celsius. The next day extract the genomic DNA with phenol-chloroform using standard procedures. Amplify 100 to 200 bases pairs of DNA fragments from gels using either a gel extraction kit or directly from PCR products using a PCR SV mini kit.
To determine the gene editing efficiency by bacterial colony sequencing, use a TOPO-TA cloning kit to ligate the purified PCR products into a PCR4-TOPO vector. Randomly pick up bacterial clones and sequence the inserts using a universal sequencing primer provided by the TOPO-TA cloning kit. To determine the gene editing efficiency by deep sequencing, send the previously obtained purified PCR products for CRISPR amplicon sequencing in a DNA sequencing core.
In this study a tube electroporation method is effectively used to deliver CRISPR/Cas9 RNP and ssODNs to rabbit and human cells, leading to robust, precise gene editing. First, C231Y and Q235X mutations are produced in the IL2RG gene, and the R150G mutation is produced in the SPR gene in rabbit fibroblast cells. The specific sgRNA designs and the gene editing rates determined by bacterial TA cloning are shown here.
The IL2RG C231 locus, out of the 28 clones sequenced, one carries the precise C231Y mutation, four carry insertion or deletion mutations, and the remaining 23 are wild-type. At the IL2RG Q235 locus, out of the 27 clones sequenced, two carry the precise Q235X mutation, three carry an indel mutation, and the remaining are wild-type. At the SPG R150 locus, of the 20 clones sequenced, five carry the precise R150 gene mutation, 10 carry indel mutations, and the remaining are wild-type.
Tube electroporation is then used to deliver Cas9 RNP and ssODNs to human iPSCs and target clinically relevant loci in EGFR, Mybpc3, and HBB genes. At the EGFR locus, 15.68%of alleles carry the precise point mutations, 22.75%carry indel mutations, and the remaining 61.57%are wild-type. At the Mybpc3 locus, 37.92%carry the precise four base pair TGAA deletion, 2.24%carry indel mutations, and the remaining 59.84%are wild type.
At the HBB locus, 11.5%carry the precise E6V mutation, 35.4%carry indel mutations, and the remaining 65%are wild-type. Cas9 RNP is the preferred form of Cas9 due to its smaller size in comparison to plasmid DNA. The tube electroporation is an effective method to deliver Cas9 RNP.
We are particularly interested in utilizing tube electroporation of Cas9 RNP in primary T cells for CAR T applications.
Overview
This article presents a protocol for CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation. This novel method enhances cellular survival and gene editing rates while being free from viruses and drugs.
Key Study Components
Area of Science
- Gene editing
- CRISPR/Cas9 technology
- Cell biology
Background
- Efficient delivery of Cas9 elements is crucial for gene editing.
- Tube electroporation is a new method that improves delivery efficiency.
- The method is applicable to various cell types, including human iPSCs and rabbit fibroblasts.
- High survival rates and gene editing efficiency are essential for clinical applications.
Purpose of Study
- To demonstrate a novel tube electroporation method for gene editing.
- To achieve high editing rates in mammalian cells.
- To provide a protocol that is free from viral and drug interventions.
Methods Used
- Preparation of human iPSCs and rabbit fibroblast cultures.
- Transfection using Cas9 RNP and donor oligonucleotides.
- Electroporation using a specific tube method.
- Assessment of gene editing efficiency through sequencing.
Main Results
- Successful delivery of CRISPR/Cas9 components to various cell types.
- High rates of precise gene editing in targeted loci.
- Demonstrated mutations in IL2RG and SPR genes in rabbit fibroblasts.
- Editing efficiency in human iPSCs at clinically relevant loci.
Conclusions
- Tube electroporation is an effective method for gene editing.
- The method allows for high cellular survival and editing rates.
- It is suitable for clinical applications due to its efficiency and safety.
Frequently Asked Questions
Executive Industry Relevance
Efficient and safe delivery of CRISPR/Cas9 components is a critical bottleneck in therapeutic gene editing development. Tube electroporation enables high-precision editing in clinically relevant human iPSCs and primary cells without viral vectors or drug-based transfection reagents, reducing immunogenicity risks and supporting scalable, GMP-compatible workflows. This approach directly addresses the need for mechanistic de-risking in early-stage genome editing programs by providing quantitative, reproducible editing outcomes across disease-relevant loci.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables functional validation of disease-associated mutations in IL2RG, SPR, EGFR, Mybpc3, and HBB genes to confirm pathogenic mechanisms.
- Operational Value: Supports rapid generation of isogenic cell lines for target de-risking and pathway interrogation in rabbit fibroblasts and human iPSCs.
Screening & Assay Development
- Scientific Value: Produces quantifiable precise editing rates (3.57%-37.92%) via bacterial TA cloning and deep sequencing, enabling dose-response and condition comparison.
- Operational Value: Delivers standardized, reproducible editing outcomes across cell types, supporting assay readiness for CRISPR-based screening platforms.
Translational & Preclinical Research
- Scientific Value: Generates clinically relevant disease models (e.g., HBB E6V for sickle cell, EGFR mutations) for preclinical efficacy and safety testing.
- Operational Value: Enables continuity from discovery to preclinical validation using the same editing method, reducing variability in translational handoff.
Pipeline & Workflow Integration
Tube electroporation fits within the discovery-to-preclinical continuum by enabling precise genome editing for target validation, assay development, and disease model generation.
- Discovery Biology: Supports hypothesis testing through precise introduction of disease-causing mutations to clarify gene function and pathway involvement.
- Screening: Delivers quantitative editing readouts that allow comparison of gRNA efficacy, donor template design, and electroporation conditions.
- Analytics: Provides measurable outputs (precise mutation rates, indel frequencies) via sequencing to inform go/no-go decisions in target validation.
- Translational Research: Generates isogenic human iPSC models with clinically relevant mutations for preclinical mechanistic and toxicity studies.
- Enterprise Reuse: Establishes a reusable, non-viral editing platform applicable across multiple cell types and therapeutic areas.
Operational & Enterprise Impact
- Scientific Value: High precision editing reduces mechanistic ambiguity in target validation and phenotypic screening.
- Operational Value: Virus- and drug-free delivery improves scalability, reduces regulatory burden, and supports consistent manufacturing.
- Strategic Value: Enables data-driven target prioritization by linking editing efficiency to phenotypic outcomes.
- Portfolio Impact: Facilitates risk-adjusted advancement of genome editing programs through validated, reproducible disease models.
Implementation Considerations
- Requires expertise in primary cell culture, iPSC handling, and electroporation optimization.
- Dependent on access to tube electroporation systems and compatible buffers.
- Necessitates standardized gRNA and ssODN design and validation protocols.
- Requires sequencing capacity (clonal or deep sequencing) for editing efficiency assessment.
- Performance may vary across primary cell types, necessitating pilot optimization.
