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TransIT-X2® Dynamic Delivery System

Experience the transfection breakthrough. Achieve superior transfections with an innovative polymeric system that efficiently delivers both DNA and RNA out of the endosome and into the cytoplasm overcoming a critical barrier to nucleic acid delivery. The TransIT-X2® Dynamic Delivery System gives researchers

 Efficiency – Exceptional Broad Spectrum Transfection

 Versatility – Cutting Edge Delivery of Plasmid DNA and small RNAs (siRNA, miRNA and CRISPR guide RNA)

 Technology – Novel, Non-Liposomal, Polymeric Delivery

Free sample available for evaluation! Please contact Genomax for sample request. 




TransIT-X2® for CRISPR/Cas9 genome editing

BACKGROUND

Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system that protects against invading viral and plasmid DNA. In Type II CRISPR systems, CRISPR RNAs (crRNAs) function with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) proteins to introduce double-stranded breaks in target DNA. Target cleavage by Cas9 requires base-pairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA (See figure CRISPR/Cas9 Genome Editing). Target recognition is facilitated by the presence of a short sequence called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG.

EDITING MAMMALIAN GENOMES WITH CRISPR

The bacterial CRISPR/Cas9 system has been adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. The Cas9 endonuclease can be programed by a dual RNA (crRNA and tracrRNA), or the core components of these RNAs can also be combined into a single hybrid guide RNA. Once the Cas9 has cleaved the target DNA, two endogenous repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR), are triggered in response to the DNA break. The features of these DNA break repair pathways can be exploited to generate gene knock-outs or introduce defined modifications at the site of cleavage. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. HDR requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence flanked on either side by sequences bearing homology to the target.

ADVANTAGES

The simplicity of using of a noncoding RNA guide to target DNA for site-specific cleavage provides a distinct advantage over alternative genome editing technologies such as ZFNs and TALENs. Using the CRISPR/Cas9 strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of DNA-binding proteins.

CRISPR/Cas9 Genome Editing. The Cas9 endonuclease (blue) is targeted to DNA by a guide RNA which can be supplied as a two-part system consisting of crRNA and tracrRNA or as a single guide RNA, where the crRNA and tracrRNA are connected by a linker (dotted line). Target recognition is facilitated by the protospacer-adjacent motif (PAM). Cleavage occurs on both strands (scissors) 3 bp upstream of the PAM.



Multiple Genomic Alterations are Possible Following Cleavage of Target DNA by Cas9. Variable length insertions and/or deletions (indels) can result near the DNA break due to mistakes in DNA repair by the endogenous non-homologous end joining (NHEJ) pathway. These indels frequently result in disruption of gene function. Alternatively, by supplying a DNA repair template, researchers can leverage the homology-directed repair (HDR) pathway to create defined deletions, insertions or modifications.

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