IDT Alt-R CRISPR-Cas9 System

Integrated DNA Technologies (IDT) introduces the Alt-R CRISPR-Cas9 System, based on the naturally occurring S. pyogenes CRISPR RNA system. The product is said to improve genome editing potency due to research-optimised 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 is a suitable 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 sgRNAs by chemical synthesis is also difficult and costly due to their length.

IDT carried out extensive research on optimising the native bacterial system of S. pyogenes, which uses a two-part crRNA:tracrRNA complex to direct Cas9 cleavage. The company 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.