CRISPR Cas9-D10A Nickase mRNA

Description

• General description: CRISPR Cas9-D10A Nickase mRNA generated through in-vitro transcription which has been capped and polyA-tailed. This mRNA should be used in conjunction with two purified guideRNAs. The use of RNA vs. plasmid constructs avoids complications with promoter-embryo compatibility as well as the possibility of random integration of nuclease and gRNA-expressing plasmids into the host animal genome.
• Application: Functional Genomics/Transgenic Applications Creation of gene knockouts in transgenic applications Creation of knock-in animals with promoters, fusion tags or reporters integrated into endogenous genes
• Features and Benefits: RNA ready to use in transgenic applications. Must be used in conjunction with two purified guideRNA sequences to mediate a site specific doubel strand break in the DNA. Recent evidence indicates off-targeting by CRISPR endonucleases is a significant concern. To address this problem, Sigma has developed paired nickase technology to expand CRISPR DNA recognition tracts to lengths similar to those of ZFNs and TALENs.  Our experience suggests that paired nickases based on the Cas9-D10A mutant are most reliable. Our development work also suggests that the critical factor in producing active paired nickases is the positioning of gRNAs in a 5′-to-5′ orientation. For RNA based delivery of paired CRISPR nickases, we recommend the use of a three part system comprised of the Cas9-D10A mRNA and two T7 generated guideRNA sequences. To simplify the use of paired nickases, Sigma has on-line tools which allow access to a library of pre-designed paired nickases. Please check Sigma′s on-line CRISPR product offerings for the latest design sets.
• Components: 1 vial containing 25ug of Cas9-D10A Nickase mRNA.Please note, product does not contain guideRNA sequence. This must be purchased separately through the Custom CRISPR product tab.
• Principle: CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a single gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB.
• Physical form: Sigma Cas9-D10A Nickase mRNA is supplied at concentrations of 500 ng/ul (50 ul Cas9-D10A mRNA, capped and polyA-tailed)
• Preparation Note: While Sigma implements the same high quality RNA synthesis procedures that have been successful in many mRNA-based ZFN transgenic projects, we highly recommend centrifuging your final, concentration-adjusted CRISPR RNA samples immediately prior to microinjection to ensure all particulate material has been removed which may result in clogging of microinjection needles.
• Other Notes: Must be used in conjunction with the RNA format of two guideRNA sequences in order to mediate a double strand break in the DNA. While Sigma CRISPR RNA was not developed for robust application to cell culture, we have seen some success in detecting CEL-I activity using RNA-only formats for both CRISPR nucleases and paired nickases.  RNA-only delivery formats may be favorable for cell types which are sensitive to double stranded DNA such a dendritic cells or when promoter-cell incompatibilities exist.Typical microinjection concentrations used in the literature are in the ranges of 20-200 ng/ul for Cas9-D10A Nickase mRNA and 10-50 ng/ul for gRNA.
• Legal Information: CRISPR Use License Agreement