Figure 1. A CRISPR-Cas9 targeted double-strand break. Cas9-mediated cleavage occurs on both strands of the DNA, three base pairs upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3’ end of the target sequence. The specificity is supplied by the guide RNA (gRNA), and changing the target only requires a change in the design of the sequence encoding the gRNA. After the gRNA unit has guided the Cas9 nuclease to a specific genomic locus, the Cas9 protein induces a double-strand break at the specific genomic target sequence.
The CRISPR-Cas9 system for genome editing
The transformative CRISPR-Cas9 technology is revolutionizing the field of genome editing. Derived from components of an adaptive immune system in bacteria, the CRISPR-Cas9 system enables targeted gene cleavage and gene editing in a wide variety of eukaryotic cells. Because the specificity of the endonuclease cleavage is guided by RNA sequences, editing can be directed to virtually any genomic locus simply by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to the target cell. The CRISPRCas9 system has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and the development of disease-resistant transgenic plants.
The CRISPR-Cas9 system derives its specificity from a short, noncoding guide RNA (gRNA) that has two molecular components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating CRISPR RNA (tracrRNA). The gRNA guides the Cas9 protein to a specific genomic locus via base pairing with the target sequence (Figure 1). Upon binding to the target sequence, the Cas9 protein induces a specific double-strand break. Following DNA cleavage, the break is repaired by cellular repair machinery through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. With target specificity defined by a very short RNA sequence coupled with an efficient endonuclease activity, the CRISPR-Cas9 system greatly simplifies directed genome editing.
Choose the right CRISPR-Cas9 delivery method
Several strategies are available for delivering Cas9 protein to target cells, and this flexibility is one of the key advantages when using CRISPR-Cas9 genome editing technology in different experimental systems (Figure 2). Advances in DNA, mRNA, and protein delivery methods have significantly streamlined the process, making the introduction of Cas9 more efficient and with minimal off-target effects. Thermo Fisher Scientific offers four formats for CRISPR-Cas9 delivery: Invitrogen™ GeneArt™ CRISPR Nuclease Vector (DNA), GeneArt™ CRISPR Nuclease mRNA (mRNA), GeneArt™ Platinum™ Cas9 Nuclease (protein) (Figure 2), and CRISPR library services (see "Apply CRISPR-Cas9 gene editing to high-throughput screening: LentiArray CRISPR libraries"). Based on the cell type and application, the most effective delivery format can be chosen and then paired with optimal cell culture reagents and analysis tools.
Figure 2. Options for efficient CRISPR-Cas9 delivery. In the DNA delivery format, the CRISPR DNA vector enters the cell and translocates to the nucleus, where the Cas9 mRNA and gRNA are transcribed. Translated in the cytoplasm, the Cas9 protein combines with the gRNA to form a ribonucleoprotein (RNP) complex that then enters the nucleus for targeted gene editing. In the RNA delivery format, the Cas9 mRNA and gRNA are cotransfected into the cell cytoplasm, where the mRNA is translated to produce functional Cas9 protein. The Cas9-gRNA (RNP) protein delivery format streamlines cell engineering by eliminating transcription and translation in the cell and produces the highest cleavage efficiencies in our labs. With the RNP format there is no requirement for a specific promoter, nor concern over random integration into the genome; the Cas9 RNP complex can act immediately after it enters the cell, since transcription and translation are not required. Moreover, the complex is rapidly cleared from the cell, minimizing the chance of off-target cleavage events when compared to vector-based systems.
Monitor the genome editing process from start to finish
Whichever CRISPR-Cas9 delivery strategy you choose, it is important to carefully monitor the entire genome editing process to validate that Cas9 protein has been successfully incorporated into cells and that the target knockout or mutation has been accurately implemented. This monitoring can be broken down into four categories:
Cell culture. The starting point for genome editing is healthy cells. Performing cell health assays prior to using the CRISPR-Cas9 system can serve as an important quality control step and help to avoid wasting time and reagents. Tests for viability, apoptosis, and stress responses should be a routine part of cell growth and can provide information to optimize experimental conditions to produce the most robust cells.
Genome editing. Immunochemical assays such as western blots can effectively measure the presence of Cas9 in cells. Figure 3 shows that the accumulation of Cas9 protein varies considerably depending on the choice of delivery method (plasmid, mRNA, or protein). Together with immunocytochemistry, antibiotic selection and gene expression 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.
Monitoring the efficiency of genome editing. When using genome editing tools—such as CRISPR-Cas9, TAL effectors, or zinc finger nucleases—to obtain targeted mutations, you need to determine the efficiency with which these nucleases cleave the target sequence, prior to continuing with labor-intensive and expensive experiments. The Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit provides a simple and reliable assay for the cleavage efficiency of genome editing tools at a given locus. In this assay, a sample of the edited cell population is used as a direct PCR template for amplification with primers specific to the targeted region. The PCR product is then denatured and reannealed to produce heteroduplex mismatches where double-strand breaks have occurred, resulting in insertion/deletion (indel) introduction. These mismatches are recognized and cleaved by the detection enzyme, and the cleavage is easily detectable and quantifiable by gel analysis.
Cell phenotyping. The CRISPR-Cas9 system is routinely used for knockout, knock-in, or modulation of gene expression, and the primary on-target effects can be measured using cell analysis techniques; western blotting, flow cytometry, and fluorescence microscopy are often used to view changes to protein expression or structure in a cell population. Flow cytometry provides the throughput for multiparameter analysis on vast numbers of individual cells. Cell imaging (Figure 4) allows for direct analysis of changes in protein expression, compartmentalization, and cell morphology; high-content analysis (HCA) provides automation for the imaging process coupled with quantitative rigor.
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 (off-target phenotyping). HCA is particularly suited to this type of multiparameter investigation (Figure 5).
Figure 3. Western blot detection of Cas9 accumulation over time in cells transfected with Cas9-expressing plasmid DNA, Cas9 mRNA, or Cas9 protein. HEK293FT cells were transfected with Cas9 plasmid DNA, mRNA, or protein and then harvested at indicated times for western blot analysis. Proteins in the cell lysates were separated on an Invitrogen™ NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gel, transferred to a PVDF membrane using the Invitrogen™ iBlot™ 2 Gel Transfer Device, and incubated with an anti-Cas9 mouse monoclonal antibody at 1:3,000 dilution and an HRP-conjugated rabbit anti–mouse IgG antibody at 1:2,000. The membrane was developed using Thermo Scientific™ Pierce™ ECL Western Blotting Substrate. Reprinted with permission from Liang X, Potter J, Kumar S et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53.
Figure 4. Absence of LC3B in CRISPR-Cas9–edited HAP1 cells after chloroquine treatment. HAP1 cells were modified using CRISPR-Cas9 gene editing to knock out the ATG5 gene. After chloroquine treatment, which normally causes LC3-containing autophagosomes to accumulate, edited cells (right panel) show the expected absence of LC3B-positive puncta, whereas wild-type cells (left panel) show an increase in LC3B accumulation. Cells were labeled with rabbit anti-LC3B antibody followed by Invitrogen™ Alexa Fluor™ 647 goat anti–rabbit IgG antibody (red) and counterstained with Hoechst™ 33342 (blue). Images were acquired on a Thermo Scientific™ CellInsight™ CX5 High-Content Screening (HCS) Platform..
Figure 5. Rapid analysis of various cell health parameters using a high-content analysis (HCA) platform. Wild-type and CRISPR-edited HAP1 cells were analyzed using the Thermo Scientific™ CellInsight™ CX5 High-Content Screening Platform for (A) apoptosis, using CellEvent™ Caspase-3/7 Green Detection Reagent, (B) oxidative stress, using CellROX™ Green Reagent, (C) protein degradation, with the Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit, and (D) protein synthesis, using the Click-iT™ Plus OPP Alexa Fluor™ 488 Protein Synthesis Assay Kit.
Resources to help you get started
Thermo Fisher Scientific offers a wide range of reagents, kits, and tools to support your genome editing experiments (Table 1). In addition to our state-of-the-art online Invitrogen™ CRISPR Search and Design Tool, we offer several different Cas9 delivery systems as well as cell culture reagents and cell analysis tools that can be matched to your experimental system. Our suite of genome editing products is continually expanding to include the entire cell engineering workflow, from reagents for cell culture, transfection, and sample preparation to kits for genome modification and for detection and analysis of known genetic variants.
Table 1. Online CRISPR-Cas9 resources from Thermo Fisher Scientific.
For Research Use Only. Not for use in diagnostic procedures.