Essential tools to optimize your genome editing experiments and analyze your results
Whichever genome editing strategy you use, careful monitoring of the process will help you generate robust and reliable results. Start with accurate cell counts and viability determinations before you commit more expensive resources. Select monitoring tools to optimize the editing steps based on your biological models. Then analyze your new cell phenotypes for targeted and off-target effects. See the examples below and follow the links for more information.
The starting point for gene editing is healthy cells. Testing for viability, apoptosis or stress responses should be a routine process. Count cells to optimize your experimental conditions and check their robustness. Cell health assays prior to editing can be a stringent QC step to avoid wasted time and reagents.
Transmitted light imaging for cell counting
Routine testing for cell health is often just viability and morphology assessment performed along with cell counting using microscopy with transmitted light illumination.
Figure 1. Cell counting using transmitted light imaging. (A) A549 (B) and MDA-MB-231 cells imaged with transmitted light at 20x magnification using the Invitrogen EVOS XL Core Imaging System. The EVOS XL Core system fits in the cell culture hood for convenient monitoring of cell health during culture and handling.
Automated cell counting
Counting viable cells can be automated using the Invitrogen Countess II FL Automated Cell Counter.
Figure 2. Cell counting with the Countess II FL Automated Cell Counter. U2OS cells were stained using calcein AM and EthD-1 as supplied in the Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit. The cell suspension was then evaluated using the GFP and Texas Red dye (TxRed) Light Cubes in the Countess II FL Automated Cell Counter. The Countess II FL counter reports total cell concentration as well as live and dead cell counts.
Quick and easy cell viability assays are readily performed using a fluorescence microscope, cell counter or flow cytometer with Invitrogen reagents. ReadyProbes cell viability assays are ready-to-use imaging solutions with a choice of one- or two-color assays. Each reagent is stable at room temperature and is dispensed from a dropper bottle.
Figure 1. Cell viability determination. Live Jurkat cells, or Jurkat cells treated with 0.1% Triton detergent, were mixed and stained using the (A)Invitrogen ReadyProbes Cell Viability Imaging Kit (Blue/Green) or (B)Invitrogen ReadyProbes Cell Viability Imaging Kit (Blue/Red). Cells were imaged and analyzed on the Invitrogen EVOS FL Auto Cell Imaging System. NucBlue Live stains all cells, while (A) NucGreen Dead and (B) propidium iodide stain only dead cells respectively.
A rapid assay for apoptosis is to simply add Invitrogen CellEvent Caspase-3/7 Green Detection Reagent to cells, incubate 30 minutes, and visualize. Apoptotic cells with activated caspase-3/7 will have bright green nuclei, while cells without activated caspase-3/7 will have minimal fluorescence. Invitrogen SYTOX Red Dead Cell Stain can be incorporated to measure dead cells.
Apoptosis measurements on an automated cell counter
Figure 1. Apoptotic and dead cells counted on a Countess II FL Automated Cell Counter. HeLa cells were labeled with 1:400 CellEvent Caspase-3/7 Green Detection Reagent to identify apoptotic cells, and then stained with 1:1,000 SYTOX Red Dead Cell Stain and incubated at room temperature for 30 minutes to label all dead cells. Cells were counted using a Countess II FL Automated Cell Counter.
Apoptosis measurements on a flow cytometer
Figure 2. Apoptotic and dead cells counted using flow cytometry. Jurkat cells (T-cell leukemia, human) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours before labeling with the reagents in the Invitrogen CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit. Stained samples were analyzed on the Invitrogen Attune NxT Flow Cytometer equipped with a 488-nm laser. Fluorescence emission was collected using a 530/30 BP filter for CellEvent Caspase-3/7 Green Detection Reagent and a 690/50 BP filter for Invitrogen SYTOX AADvanced Dead Cell Stain Kit, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A = apoptotic cells, L = live cells, N = necrotic cells.
Antibiotic selection, gene expression, and immunocytochemistry assays are frequently used to monitor the assembly of CRISPR components for gene editing in the cell. Fluorescent protein expression can be measured directly, and when antibiotic selection is used to identify transfected cells, viability assays can be used to monitor the selection process.
Measuring lentivirus delivery using automated cell counting
Figure 1. GFP expression measured using the Countess II FL Automated Cell Counter. U2OS cells expressing the Cas9 protein were transduced with Invitrogen LentiArray Positive Ctrl gRNA (HPRT-GFP) and Invitrogen LentiArray Negative Ctrl gRNA (Scrambled-GFP) at MOIs of 1 and 2. Two days later, cells were counted and measured for GFP on the Countess II FL Automated Cell Counter. Measurements showed the percentage of cells positive for GFP, and indicated the percentage of transduced cells expressing the GFP and puromycin resistance gene (2A and 2B).
Measuring lentivirus delivery using transmitted light imaging
Figure 2. Antibiotic selection using the EVOS XL Core Imaging System. Cells transduced with Invitrogen GeneArt Lentiviral CRISPR Positive Ctrl particles (HPRT-GFP) show normal viability (A) before treatment. After 4 days under puromycin selection (B), cells are clumped and stressed in response to antibiotic.
Measuring lentivirus delivery using flow cytometry
Figure 3. Measuring transfection efficiency in 293T cells using flow cytometry. The Invitrogen GeneArt CRISPR Nuclease Vector with OFP Reporter Kit uses expression of an orange fluorescent protein (OFP) to label transfected cells. Transfection efficiency was measured in 293T cells using the Attune NxT Flow Cytometer, and the data shows >90% OFP-positive cells in transfected samples.
Measuring Cas9 in transfected cells using western blotting offers a way to assay transfection efficiency and optimize the editing process by measuring protein expression in the total cell population.
Western blotting to assess Cas9 delivery methods
Figure 1. Western blot detection of Cas9 accumulation over time in plasmid DNA, mRNA, and protein transfected cells. HEK293FT cells were transfected with Cas9 plasmid DNA, mRNA, or protein. Cells were harvested at indicated times to perform western blot analysis. Proteins in the cell lysate were separated on an Invitrogen NuPAGE 4–12% Bis-Tris gel, transferred to PVDF membrane using the Invitrogen iBlot 2 Gel Transfer Device, incubated with mouse monoclonal Cas9 antibody at 1:3,000 dilution and rabbit anti-mouse HRP conjugated secondary antibody at 1:2,000. The membrane was developed using Thermo Scientific Pierce ECL substrate.
Xiquan Liang et. Al., Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology, Volume 208, Pages 44–53.
Western blot detection of Cas9
Figure 2. Western blot detection of Cas9. Western blotting was performed on protein extracts from HEK293 cells transfected with Myc-tagged Cas9 using a CRISPR-Cas9 monoclonal antibody at different dilutions. The positions of the mass marker bands are shown on the left; the position of the Myc-tagged Cas9 protein is indicated (arrow).
Measuring Cas9 using immunocytochemistry
Immunocytochemistry is a standard technique for identifying specific proteins in the cell. Using an anti-Cas9 primary antibody and a labeled secondary antibody, Cas9 expression can be detected on a cell-by-cell basis with fluorescence imaging.
Figure 3. Monitoring Cas9 delivery. (A) U2OS cells and (B) U2OS-Cas 9 cells were treated with CRISPR-Cas9 monoclonal antibody, then stained with a goat anti-mouse Alexa Fluor 594 conjugate and Hoechst 33342. Red punctate staining in the cytoplasm shows the presence of Cas9. Cells were visualized using an EVOS FL Auto Cell Imaging System.
When antibiotic selection is used to identify transfected cells, the Countess II FL Automated Cell Counter with a viability stain such as trypan blue gives a quick readout of viability to allow easy comparisons between treatments.
Figure 1. U2OS-Cas9 cells with gRNA. (2A) Cells transduced with GeneArt Lentiviral CRISPR Positive Ctrl particles (LentiArray Positive Ctrl gRNA (HPRT-GFP)) show normal viability after 4 days under puromycin selection. (2B) After 14 days under puromycin selection, cells transduced with GeneArt Lentiviral CRISPR Negative Ctrl particles (LentiArray Negative Ctrl gRNA (Scrambled-GFP)) and GeneArt Lentiviral CRISPR Positive Ctrl particles (LentiArray Positive Ctrl gRNA (HPRT-GFP)) show viability levels similar to U2OS Cas9 stable cells not under puromycin selection. Cells were counted using a Countess II FL Automated Cell Counter.
The functionality of nuclease cleavage in cultured cells can be screened in a quantitative assay using cell lysates and a gel-based readout, or via a fluorescent readout in live or fixed cells. In the fluorescence-based assay, fluorescent protein expression can be employed as an enriching tool via cell sorting. Or, surface proteins can be assayed using Invitrogen Dynabeads CD4 magnetic beads.
Gel analysis of cell lysates to confirm cleavage
Figure 1. CRISPR-Cas9–mediated cleavage efficiency. Gel image of a cleavage assay using the Invitrogen GeneArt Genomic Cleavage Detection Kit for the HPRT locus. (A) Results using the GeneArt CRISPR Nuclease OFP Vector expressing HPRT-specific CRISPR RNA. (B) Results obtained using the GeneArt CRISPR Nuclease CD4 Vector expressing HPRT-specific CRISPR RNA. Following transfection into HeLa cells, triplicate cleavage assays were performed, and the percentage of indels was calculated.
Fluorescence-based cell sorting assay to confirm cleavage
The Invitrogen GeneArt Genomic Cleavage Selection Kit is used for detecting the functionality of engineered nucleases in transfected cells. Active nuclease activity results in the expression of Orange Fluorescent Protein (OFP) and CD4 surface antigens. These markers can also be used to enrich for modified cells using fluorescence-activated cell sorting (FACS) or Dynabeads CD4 magnetic beads.
Figure 2. Nuclease expression in 293FT cells using the GeneArt Genomic Cleavage Selection Kit. Transfected cells were stained with anti-CD4 Alexa Fluor 488 antibody and analyzed using the Attune NxT Flow Cytometer to count nuclease-modified cells. The CD4 reporters can be used for enrichment of the nuclease-modified cells using fluorescence-activated cell sorting (FACS) or CD4 antibody-conjugated Dynabeads magnetic beads.
Analysis of cleavage efficiency using Sanger sequencing
Efficiency of nuclease cleavage can be determined using Sanger sequencing. In one approach, lysate from treated cells can be sequenced using standard methods. The resulting traces, showing a mixed sequence where the edit occurred (Figure 3A), can be analyzed using third-party software such as Tracking of Indels by Decomposition (TIDE, Brinkman, EK et al. (2014)). Another method for determining nuclease cleavage efficiency is to subclone the amplicon from treated cells into plasmids, and Sanger sequence the insert in individual bacterial subclones. Counting the number of subclones with a successful edit gives a measure of the efficiency of the editing reaction, as well as visualizing the type of sequences that resulted from the edit (see application note: Using Sanger sequencing to facilitate CRISPR- and TALEN-mediated genome editing workflows).
Figure 3. Use of Sanger sequencing for determining editing efficiency. (A) A mixed population of HEK293 cells with a genome edit event at the human HPRT locus was analyzed using SeqScanner 2 software. Note the position of the edit, indicated with the red arrow. (B) Analysis of the sequence trace using TIDE software. The fraction of cell predicted to have -1, -2, -3 etc deletions, as well as the overall predicted editing efficiency, is shown.
The HPRT gene encodes a transferase involved in purine nucleotide synthesis, and expression of the gene results in the incorporation of 6-thioguanine (6-TG) into DNA, which inhibits cell viability. Growth medium containing 6-TG is used as a selection tool for gene knockout, since cells expressing HPRT lose viability and can be screened via cell morphology using fluorescence microscopy, or in a Countess II FL Automated Cell Counter assay with trypan blue. Scramble-GFP serves as a control construct with no editing of HPRT.
HPRT viability screening using fluorescence microscopy
Figure 1. Negative selection with HPRT.(A) After 6 days under selection with 6-thioguanine, Scramble-GFP samples show clumped morphology and decreased cell viability, indicating 6-TG is being incorporated into the DNA instead of purine nucleotides. (B) HPRT-GFP samples show normal cell viability/morphology, indicating the HPRT gene has been knocked out resulting in 6-TG not being incorporated into the DNA and normal cell growth. Cells were visualized using the EVOS FL Auto Imaging System.
HPRT viability screening using automated cell counting
Figure 2. Negative selection with HPRT. (3A) After 6 days under selection with 0.6 µg/mL 6-thioguanine, Scramble-GFP samples show decreased cell viability, indicating 6-TG is being incorporated into the DNA instead of purine nucleotides. (3B) HPRT-GFP samples show normal cell viability, indicating the HPRT gene has been knocked out resulting in 6-TG not being incorporated into the DNA and cell survival. Cells were counted using a Countess II FL Automated Cell Counter.
CRISPR is routinely used for knockout, knock-in, or modulation of gene expression, and the effects can be measured using cell analysis techniques. Western blotting is used to view changes to protein expression in a cell population; flow cytometry provides the throughput for multiparameter analysis on vast numbers of individual cells. Imaging allows for direct analysis of changes in protein expression, compartmentalization, and cell morphology, while high-content analysis (HCA) provides automation for the imaging process with quantitative rigor.
These experiments use CRISPR knockout of ATG5 to illustrate the effects of protein modulation in the autophagy pathway.
Western blotting to assess target expression
Western blotting provides the means to quantify a phenotypic change such as protein expression in a population of cells.
Figure 1. CRISPR-edited Hap1 cells accumulate LC3B after chloroquine treatment. LC3B can be measured using quantitative microscopy or western blotting.
Qualitative analysis using fluorescence microscopy
Microscopy and flow cytometry–based techniques measure responses at the individual cell level, whereas western blotting measures a specific protein extracted from a population of cells.
Figure 2. Measuring autophagy in HeLa cells using anti-LC3B. HeLa cells treated with chloroquine were fixed and permeabilized, and subsequently labeled with an anti-LC3B antibody followed by a goat anti-rabbit Alexa Fluor 488 conjugate (green). Cells were counterstained with Hoechst 33342 (blue) and imaged using an EVOS FL Auto Imaging System for qualitative analysis.
High-content analysis (HCA) of cellular changes
Figure 3. Automatic quantitation of autophagy using HCA. A Thermo Scientific CellInsight CX7 platform was used to identify and count LC3B granules in CRISPR-edited HAP1 cells. Cells were labeled with HCS NuclearMask Blue, HCS CellMask Deep Red, and an anti-LC3B antibody followed by Invitrogen Alexa Fluor 647 goat anti-rabbit antibody. Automated analysis using Thermo Scientific HCS Studio 3.0 identified nuclei (blue overlay) cell perimeter (green outline) and quantified LC3B granules (red overlay in fluorescence image and in bar chart).
With modulation of any cellular signaling pathway comes the risk of proximal and distal consequences. It is important to track your targeted protein and also monitor the impact on other aspects of cell health and behavior. High-content screening (HCS) is particularly suited to this type of multiparameter investigation, and Invitrogen reagents provide the breadth of tools for interrogating cell health and behavior
Figure 1. Rapid analysis of various cell health parameters using the Thermo Scientific CellInsight CX5 platform. Wild-type and CRISPR-edited Hap-1 cells were analyzed using the CellInsight CX5 HCS Platform for (A) apoptosis using CellEvent Caspase-3/7 Green Detection Reagent, (B) oxidative stress using Invitrogen CellROX reagents, (C) protein degradation using Invitrogen LysoTracker reagents, and (D) protein synthesis using an Invitrogen Click-iT OPP assay kit.
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