Insightful harnessing of the natural crRNA:tracrRNA complex with the new Alt-R™ CRISPR-Cas9 System
CORALVILLE, IA (October 28, 2015) – Integrated DNA Technologies (IDT), introduces a new approach to CRISPR—its innovative Alt-R™ CRISPR-Cas9 System, based on the naturally occurring S. pyogenes CRISPR RNA system. The Alt-R CRISPR-Cas9 System greatly improves genome editing potency thanks to research-optimized crRNAs (CRISPR RNA) and tracrRNAs (trans-activating crRNA). Along with improved potency and safety, the system saves time by providing easy, ready-to-use RNA reagents that also reduce cell toxicity by avoiding activation of cellular innate immune responses.
The CRISPR-Cas9 system has become a leading tool for modifying genomes of organisms from E. coli to humans. Chimeric single-guide RNAs (sgRNAs, composed of a fusion between crRNAs and tracrRNAs) are commonly cloned into plasmid vectors, and then used to express the RNA trigger in cells. However, the use of plasmid vectors to drive genome editing lacks efficacy and is suspected of causing significant off-target effects, while sgRNAs obtained by in vitro transcription can be expensive and cytotoxic through activation of the innate immune system in many mammalian cell types. Manufacture of high quality sgRNAs by chemical synthesis is also difficult and costly due to their length.
Addressing the problems of the sgRNA-based system, IDT carried out extensive research on optimizing the native bacterial system of S. pyogenes, which uses a two-part crRNA:tracrRNA complex to direct Cas9 cleavage. IDT researchers found that not only was it possible to shorten both the crRNA and tracrRNAs to 36 and 67 nucleotides respectively, but that doing so increased the on-target potency of the reaction compared with other approaches. Shortening of these RNAs enables IDT to manufacture the components as high quality, synthetic RNA oligonucleotides that elicit less toxicity and innate immune response activation in cells. Researchers can also benefit from a safe, fast and easy protocol with no viral particle preparation, in vitro transcription, or purification steps.
Dr Mark Behlke, CSO of IDT commented; “We are very excited about our new Alt-R CRISPR-Cas9 product range. For us, it was logical to reconsider the natural system, as nature is generally very good at designing highly effective solutions—sometimes a skillful nudge in the right direction is all that’s needed. Our research in this area found that shortening the natural crRNAs and tracrRNAs results in an unexpected, yet significant, improvement on current CRISPR methods. Not only is editing efficiency improved, but the shorter synthetic RNAs are more easily made using high quality, high-throughput manufacturing methods. This two-part RNA system is also particularly useful for very clean manipulations in cells, as it doesn’t leave a footprint that could cause undesired changes to the genome, like DNA-directed expressions systems can. We think this system shows great promise not only for making CRISPR much more accessible and efficient in research but also in therapeutic applications for exactly that reason.”
Researchers can find out more about the innovative new system in a webinar titled “Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Design and use” on Wednesday, November 4, 2015. Dr Garrett Rettig, a Research Scientist in Molecular Genetics at IDT who led research efforts in CRISPR genome editing methods will present the webinar at 9 am and 1 pm Central Standard Time. The presentation follows on from the recent “New RNA tools for optimized CRISPR/Cas-9 genome editing” webinar, which presented details of the research that developed the Alt-R CRISPR-Cas9 System. The new webinar will feature a detailed look at the components of the system, explain how to design and order Alt-R CRISPR crRNAs, and provide a step-by-step guide to the experimental process.
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