The goal of the Genome Editing Centre at Utrecht University is to assist researchers with their gene editing projects. We provide advice, protocols and tools for gene editing of your target cell type, including recombinant Cas9 proteins and reagents for the synthesis of sgRNA.
The CRISPR/Cas system
The discovery of CRISPR/Cas9 gene editing systems has greatly accelerated the way in which we can modulate gene expression and function. The appeal of this system lies in its simplicity, in its minimal form consisting of just two components: a Cas9 nuclease and a single guide RNA (sgRNA). The sgRNA homes the complex to a specific site in the genome and cleaves double-stranded DNA, resulting in the activation of the cellular DNA repair machinery. Repair of the break through Non-Homologous End Joining (NHEJ) results in insertions and/or deletions (InDels), thereby disrupting the target locus. When, in addition, donor template DNA, with homology to the target locus, is delivered into the cell as well, the cell will use this template DNA for repair of the break via homology-directed repair (HDR), resulting in precise changes at the target site.
Variants of the Cas9 protein
Variations to the original CRISPR/Cas system have been generated to expand the way in which this system can be used in genome modulation. A single point mutation in the Cas9 gene (D10A) converts this enzyme into a nickase. Target specificity can be improved when loci are targeted with a pair of adjacent Cas9 nickases.
In addition, a nuclease-dead Cas9 (dCas9) was generated by combining mutations in the RuvC domain (D10A) and the HNH domain (H840A). The dCas9 variant can be targeted to a specific site in the genome without resultant cleavage. By fusing dCas9 with various effector domains, a host of new gene modulating tools can be created that can silence or activate genes, and alter chromatin structure or DNA methylation.
For more information, please visit us at www.uugc.nl.