CRISPR/Cas Nuclease RNA-guided Genome Editing
Sigma Aldrich is proud to offer its newest line of genome editing tools, Sigma CRISPRs, to the global research community. As the first company to commercially offer targeted genome editing technology nearly ten years ago, no one has more expertise in this field than Sigma Aldrich. This experience is especially important when it comes to crafting genome editing tools that possess the critical requirements of having specific targeting and robust cutting activity. We provide both in our new Sigma CRISPR product line, and now we put our skill into your hands with a quick and simple web-based design platform. Sigma CRISPR products can be ordered directly through the link below or browse the CRISPR content on this page to learn more about the technology.
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What is CRISPR/Cas?
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The discovery of the type II prokaryotic CRISPR “immune system” has allowed for the development for an RNA-guided genome editing tool that is simple, easy and quick to implement.
How does CRISPR/Cas work?
The CRISPR pathway was discovered in bacteria, where it functions much like an immune system against invading viruses and plasmid DNA. Short DNA sequences (spacers) from invading viruses are incorporated at CRISPR loci within the bacterial genome and serve as “memory” of previous infection. Re-infection triggers the complementary mature CRISPR RNA (crRNA) to find a matching sequence – which provides the CRISPR-associated (Cas) nuclease the specificity to form a double-strand break at specific “foreign” DNA sequences.
The Cas9 nuclease and requirements for RNA-guided genome-editing
Cas9 is the nuclease guided by the crRNA and tracrRNA (or trans-activating crRNA) to cleave specific DNA sequences (Deltcheva et al. 2011). A guide RNA (gRNA) can be designed to include a hairpin that mimics the tracrRNA-crRNA complex (Jinek et al. 2012). Binding specificity is based on the gRNA and a three nucleotide NGG sequence called the protospacer adjacent motif (PAM) sequence (Marrafﬁni and Sontheimer, 2010). For site-specific genome editing, the CRISPR/Cas9 system minimally requires the Cas 9 nuclease and the gRNA.
What are the benefits of using CRISPR/Cas9 for genome-editing?
The system is simple, as it only requires a Cas nuclease and a gRNA against the target sequence to function as a site-specific nuclease. Also, despite the bacterial evolutionary origins of the system, data demonstrates high levels of cutting activity in mammalian cells (Cong et al., 2013; Mali et al., 2013), particularly at numerous simultaneous targets, (Wang et al., 2013). In addition, the requirement for an NGG sequence makes target design simple and straightforward in genomic regions where off-targeting is not an issue. Finally, CRISPR provides researchers a fast and cost-effective genome editing tool to use for modifying the genomes of various organisms.
- Deltcheva et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602-7 (2011).
- M. M. Jinek, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).
- L. A. Marraffini, E. J. Sontheimer, Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568 (2010)
- Wang et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910-8. (2013)
- Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339(6121):819-23.
- Mali, P., et al., RNA-guided human genome engineering via Cas9. Science 2013; 339(6121):823-6.