GeneArt® CRISPR Nuclease mRNA
日期：
2015-06-01
浏览次数:
395
货号： A25640
内容及储存
Store in ultra-cold freezer (-68 to -85°C). Product may be stored short-term in freezer (-5 to -30°C).
描述
GeneArt® CRISPR Nuclease mRNA is wild type Cas9 in mRNA format for genome editing with CRISPR/Cas9 technology. Cas9 mRNA is ready to transfect and circumvents the need for time-consuming cloning steps required when using CRISPR vector systems. Cas9 mRNA is co-transfected with a custom GeneArt® CRISPR Strings™ DNA encoding a target-specific guide RNA (gRNA) that directs the Cas9 protein to the intended genome locus to create a double-stranded break. The Cas9 RNA/CRISPR Strings™ DNA combination provides a smaller payload than plasmid-based Cas9 systems for improved delivery into the cell and better genome editing efficiency. Additionally, the Cas9 mRNA can be used in multiplexing approaches with more than one GeneArt® CRISPR Strings™ DNA. Use this approach to determine which gRNA sequence works best for a particular target or edit multiple genomic loci with one transfection. GeneArt® Cas9 mRNA and CRISPR Strings™ DNA may be used for screening purposes with subsequent high specific cleavage with GeneArt® Precision TALs.GeneArt® CRISPR Strings™ DNA must be purchased separately. Download the GeneArt® CRISPR Strings™ DNA order form and submit to: GeneArtSupport@lifetech.comGeneArt® CRISPR Nuclease mRNA lets you:• Circumvent time-consuming cloning steps• Screen several CRISPR gRNA sequences at once• Avoid promoter constraints when used with T7 based CRISPR StringsGeneArt® CRISPR Strings™ DNAGeneArt® CRISPR Strings™ are custom DNA fragments designed to generate the CRISPR gRNA component of the CRISPR/Cas9 system. They are offered with either U6 or T7 promoters. CRISPR Strings™ DNA with a U6 promoter can be directly transfected into the cell with Cas9 mRNA. The U6 promoter drives expression of CRISPR gRNA for complexing with the Cas9 protein generated from the Cas9 mRNA. The use of the human U6 promoter helps ensure high expression efficiency of the gRNA. CRISPR Strings™ DNA with a T7 promoter can be in vitro transcribed into gRNA using our MEGAshortscript™ T7 Transcription Kit prior to transfection. It is then combined with the Cas9 mRNA for a complete RNA format for more difficult-to-transfect cells and elimination of promoter constraints for genome editing in a broader range of cell types.About CRISPR/Cas9The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is a prokaryotic adaptive immune system that uses an RNA-guided DNA nuclease to silence viral nucleic acids (1). In bacteria, CRISPR loci are composed of a series of repeats separated by segments of exogenous DNA (~30 bp in length) called spacers. The repeat-spacer array is transcribed as a long precursor and processed within repeat sequences to generate small crRNAs that specify the target sequences (also known as protospacers) cleaved by the CRISPR nuclease. CRISPR spacers are then used to recognize and silence exogenous genetic elements at the RNA or DNA level. Essential for cleavage is a sequence motif immediately downstream on the 3’ end of the target region, known as the protospacer-adjacent motif (PAM). The PAM is present in the target DNA, but not the crRNA that targets it.Genome EditingGenome editing involves the use of engineered nucleases in conjunction with endogenous repair mechanisms to insert, delete, or replace DNA sequences from a specific location in genomic DNA. Engineered nucleases induce a double-stranded break (DSB) at a specific location in the genome, after which endogenous repair mechanisms repair the break via non-homologous end joining (NHEJ) or homology directed repair. The type II CRISPR system has been shown to function as a gene editing tool in various organisms including mammalian cells. (2,3).The CRISPR system consists of three components: the CRISPR-associated Cas9 nuclease (a double-stranded DNA endonuclease), a target complementary CRISPR RNA (crRNA), and an auxiliary trans-activating crRNA (tracrRNA). The crRNA and tracrRNA act as a short guide RNA to target the Cas9 nuclease to specific genomic loci.In the GeneArt® CRISPR Nuclease mRNA system, crRNA and tracrRNA of the GeneArt® CRISPR Strings™ DNA are expressed together as a guide RNA that mimics the natural crRNA-tracrRNA hybrid in bacterial systems. The system is versatile and simple to use, and changing target specificity only requires a change in the design of the GeneArt® CRISPR Strings™ DNA.References1. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier E. (2012) A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337:6096, 816–821.2. Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S., Yang L., Church Church, G.M. (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology 31, 833–838.3. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:6121, 819–823.