Introduction to the CRISPR/Cas9 system

A powerful method for engineering your gene of interest

Although recently developed programmable editing tools, such as zinc finger nucleases and transcription activator-like effector nucleases, have significantly improved the capacity for precise genome modification, these techniques have limitations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology represents a significant improvement over these other next-generation genome editing tools, reaching a new level of targeting, efficiency, and ease of use. The CRISPR/Cas9 system allows for site-specific genomic targeting in virtually any organism.

The type II CRISPR/Cas system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. This DNA damage is repaired by cellular DNA repair mechanisms, either via the non-homologous end joining DNA repair pathway (NHEJ) or the homology-directed repair (HDR) pathway.

The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells, and can be used to generate gene knockouts (via insertion/deletion) or knockins (via HDR). To create gene disruptions (Figure 1), a single guide RNA (sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Cas9-induced double strand breaks are repaired via the NHEJ DNA repair pathway. The repair is error-prone, and thus insertions and deletions (INDELs) may be introduced that can disrupt gene function.

The principle of CRISPR/Cas9-mediated gene disruption. A single guide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein (1), binds to a recombinant form of Cas9 protein that has DNA endonuclease activity (2). The resulting complex will cause target-specific double-stranded DNA cleavage (3). The cleavage site will be repaired by the nonhomologous end joining (NHEJ) DNA repair pathway, an error-prone process that may result in insertions/deletions (INDELs) that may disrupt gene function (4).

CRISPR/Cas9 technology has revolutionized genome editing, allowing a previously unattainable level of genomic targeting, efficiency, and simplicity. Guide-it products further improve the usability of the CRISPR/Cas9 system by providing a streamlined method for:

• Producing sgRNAs in vitro
• Testing the cleavage efficiency of sgRNAs
• Monitoring the efficiency of genome editing in cultured cells