The CRISPR/Cas system is a genome editing technology that works with a cell’s endogenous repair mechanism. The main components of the system are Cas9, which is an endonuclease, and a guide RNA or gRNA. The gRNA consists of a target-specific crRNA sequence and a tracrRNA sequence, which is a trans-activating component of the system that allows loading of Cas9 onto the gRNA. CRISPR/CAS vectors are now available from a number of commercial sources that combine these necessary components into a single vector, simplifying the experimental setup. Once the system vector is introduced into the cell it induces targeted DNA cleavage. This double-stranded DNA break can then be repaired by the cellular repair machinery using either non-homologous end joining or a homology-directed repair mechanism, introducing the desired genomic change.
Setting up a CRISPR Experiment
The first step in setting up an experiment using the CRISPR system is to clone a 20–base pair target sequence into a CRISPR/Cas expression plasmid. A variety of web tools are available to assist in designing the experiment and checking for possible off-target effects. An easy to use CRISPR design, cloning and analysis guide is available through Life Technologies. Once the target sequence is cloned into the expression plasmid, cells are transfected and analyzed to confirm the edited genome. As with any genome modification system, one key step that should not be overlooked is establishing a robust screening mechanism to ensure that the targeted genomic change is obtained and that off-target effects are avoided. Cells can be analyzed using PCR, real-time PCR, western blots, ELISAs, sequencing, or using the newest genome editing tool GeneArt® Genomic Cleavage Detection Assay. The GeneArt® Genomic Cleavage Detection Assay, provides a simple, reliable, and rapid method to determine the nuclease cleavage efficiency at a given locus. 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 products are then denatured and re-annealed to produce heteroduplex mismatches where double-strand breaks have occurred, resulting in indel introduction. These mismatches are recognized and cleaved by the detection enzyme. This cleavage is both easily detectable and quantifiable using gel analysis.
CRISPR/Cas9-mediated cleavage efficiency. Gel image of a cleavage assay using the GeneArt® Genomic Cleavage Detection Assay 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 were calculated.
Many CRISPR/Cas system vectors include a reporter or tag. Two different reporters are available through Life Technologies for enrichment of transfected cells. OFP, orange-fluorescent protein allows for fluorescence-based tracking of transfection efficiency as well as FACS-based sorting and enrichment of cells expressing Cas9 and gRNA. The second reporter that is available is CD4, a surface antigen providing an option for magnetic bead-based sorting and enrichment of cells expressing Cas9 and gRNA using Dynabeads® CD4 magnetic beads, transfection efficiency can also be tracked using a fluorescent anti-CD4 antibody.
Applications of CRISPR Technology
CRISPR technology can be used for a variety of genome modifications including gene deletion or knockout, knock-in, and knockdown. The system can introduce defined mutations into the genome or disrupt gene function via frameshifts. It has been used in a variety of cell lines and organisms including mice, humans, zebrafish, and plants. Compared to other genome editing technologies, it is inexpensive, fast, and efficient.
Free software exists for target sequence design, and vectors are available from a number of sources. For scientists looking to bypass the vector cloning stages, a variety of full-service options are now coming to market, including ready-to-transfect pre-designed expression plasmids and custom engineered stable cell lines.
CRISPR technology can help you quickly and efficiently edit and manipulate a genomic locus in many different cell types and organisms. This technology is opening up new methods to study disease models or for drug screening, enabling the creation of more sensitive experimental models and even allowing study of disease in edited cell lines.
After relevant targets have been identified with fast and easy-to-use GeneArt. CRISPRs, the biologically relevant mutations can be precisely created with GeneArt® Precision TALs with high specificity and low off-target effect.
In this Bench Tip Video, Dr. Mike Okimoto, Chief Content Officer Biocompare, discusses CRISPR/Cas genome editing technology
Related Products from: Life Technologies