A Complete Suite of Reagents, From Cas9 Delivery Tools to Cell Function Assays

The CRISPR-Cas9 system for genome editing

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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.

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).

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.