Level0, Level1 and level2 constructs for production of genetically encoded autoluminescence in plants (Mitiouchkina etal, Nature 2020) are now available. Please check out the new "Genetically encoded Autoluminescence" section on the website to see the constructs we have available.
The Cas9 protein found in type II CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci for bacterial adaptive immunity has been repurposed as an easy-to-engineer molecular tool for genome engineering. In the engineered system Cas9 is guided to specific DNA sequences by a so-called “single guide RNA” (sgRNA). The sgRNA can be re-programmed to target almost any sequence. Therefore only two components, Cas9 and an sgRNA, are required for RNA-guided Cas9-mediated genome engineering. Multiplex editing can be achieved by delivering multiple sgRNAs for different targets to the same cell.
Cas9 contains nuclease activity and induces double-strand breaks. In 2012 and 2013 several laboratories demonstrated that a nuclear-targeted cas9, co-expressed in eukaryotic cells with a sgRNA designed to an endogenous target could create a double-strand break at a pre-determined target. The breaks were repaired by the cells’ own DNA repair mechanisms. Since these mechanisms are sometimes imperfect, this resulted in small mutations at user-defined sites.
The sgRNA guide sequence confers target specificity (see Figure 1). It’s target sequence must be followed by a “Protospacer Adjacent Motif” (PAM), consisting of NGG, therefore any 23bp sequence ending ‘GG’ can be targeted by an RNA-guided Cas9.
At TSL we use TypeII restriction enzymes to rapidly assemble multigene binary vectors containing a Cas9 cassette, one or more sgRNAs and (usually) a plant selectable marker cassette. When delivered to plants, these create double-strand breaks at the target. Please read our guide on how to assemble plasmid vectors for Cas9-mediated double-strand break induction in plant genomes.