The power of gene editing provides the ability to change the biology of a cell or whole organism, enabling the investigation of a host of difficult or previously impossible to interrogate biological processes. As more and more gene editing tools are discovered and developed, the applications for gene editing will only continue to expand. Here we use CRISPR-Cas9 editing of intracellular adhesion molecule-1 in a prostate cancer cell line to illustrate a method that incorporates enrichment and single-cell sorting to allow monocultures of edited cells to be generated in a reliable and more rapid manner.
Intracellular adhesion molecule-1 (ICAM-1) plays an important role in stabilizing cell-cell interactions and in mediating cell signaling during the immune response. It is a cell surface glycoprotein typically expressed in immune and endothelial cells and binds to integrins. ICAM-1 expression can be stimulated by interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα), which facilitates leukocyte extravasation through endothelial cells into underlying tissue. ICAM-1 expressed by respiratory epithelial cells is also the binding site for rhinovirus, the causative agent of most common colds. Additionally, ICAM-1 is associated with a variety of cancer cell types and has been shown to play both a positive and a negative role in cancer cell invasion and metastasis.
To investigate the effect of decreased ICAM-1 expression on various cell signaling pathways, we analyzed gene and protein expression in a cellular knockout of ICAM-1. We found that a reduction of ICAM-1 expression through CRISPR-mediated gene editing altered gene and protein expression in the prostate cancer cell line PC3. In addition, we demonstrate a streamlined method for CRISPR gene editing that includes enrichment and single-cell sorting flow cytometry.
After designing your gene editing strategy, the first step in the CRISPR gene editing workflow is to identify the best method for delivering the CRISPR-Cas9 system into your cells of interest, be they animal or human, tumor-derived or wild type. Transfer efficiency and subsequent cell viability are very important when considering which method to use.
We designed a knockout strategy that uses knock-in of a donor template containing the GFP and puromycin resistance genes to disrupt ICAM-1. PC3 cells were transfected using either TransFectin™ Lipid Reagent or the Gene Pulser Xcell™ Electroporation System. GFP fluorescence was used as an initial readout for successful transfection (Figure 1).
2. Enrichment and Single-Cell Isolation
Following transfection, you need to enrich for your cells of interest. This step speeds up your workflow and reduces unnecessary spending on reagents and consumables by allowing identification and expansion of only those cells that carry desired edits. Enrichment also reduces the number of passages cells undergo, ensuring healthy cells for downstream analysis.
We enriched for successfully transfected cells by sorting GFP-positive PC3 cells using the S3e™ Cell Sorter (Figure 2). After enrichment and subsequent puromycin selection, the S3e Cell Sorter was used to isolate single GFP-positive, puromycin resistant cells for subsequent expansion. The ZOE Cell Imager allowed early confirmation of successful enrichment pre- and post-puromycin treatment. Compared to traditional protocols, enriching for GFP-positive cells increased the number of viable cells available for single-cell sorting and colony growth following puromycin selection (Figure 3). This enrichment step brought cells to the single-cell sort stage approximately 30 days earlier than a protocol without enrichment (Figure 4).
3. Confirmation of Edits
Once you have enriched your cells of interest, you need to confirm that you have successfully edited your target cells before moving on to downstream assays. This can be accomplished through direct detection of edits using genomic methods or through indirect detection using cellular or proteomic methods.
Protein analysis by western blot (Figure 5) and immunocytochemistry (ICC, Figure 6) showed a complete loss of ICAM-1 expression in our edited cells.
4. Downstream Analysis
Now that you have confirmed that your cells are correctly edited, you can begin to investigate their phenotype. You may even wish to further perturb your system with a drug as part of a cell-based assay during target or lead discovery and validation.
Using PrimePCR™ Real-Time PCR Assays and Bio-Plex® Multiplex Immunoassays, we were able to show gene and protein expression of ICAM-1 in our knockout cell lines upon stimulation with TNFα, albeit to a lesser extent than in control unedited PC3 cells. We found expression changes in a number of genes and proteins related to the extracellular matrix and other cell signaling pathways (Figure 7 and data not shown).
Enrichment of transfected cells by flow cytometry saves time, reduces cell culture waste, and increases the chances of selecting and growing successfully edited colonies. Reducing the level of ICAM-1 expression in PC3 cells affects other cell signaling pathways involved in extracellular matrix remodeling and cell signaling under both basal and stimulated conditions.
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The Bio-Plex Suspension Array System includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation.
Bio-Rad’s thermal cyclers and real-time thermal cyclers are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.