CRISPR / Cas9
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system was first discovered in bacteria and provides sequence specific adaptive immunity against foreign DNA. Following its initial demonstration, scientists successfully tuned CRISPR system into a gene-editing tool enables permanent modification of targeted genes in living organisms.
How CRISPR / Cas9 works?
The CRISPR genome editing system requires a guide RNA (gRNA) and Cas9. gRNA is a short synthetic RNA guides Cas9 to the specific location in the genome sequence. The gRNA and Cas9 complex is like a pair of scissors that can cleave both strands of DNA upon recognition of particular DNA sequences. By simply redesigning the targeting sequence in the gRNA, we can change the genomic target of Cas9.
After both strands are cut, the break is repaired by endogenous DNA repair mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR). HDR uses donor DNA containing a desired sequence resulting in the correction of a disease-causing mutation. Currently, the delivery of gRNA, Cas9 together with Donor DNA is a huge challenge. GenEdit has a proprietary technology enabling efficient HDR.
CRISPR delivery vehicle
Our proprietary CRISPR-Nanoparticle can directly deliver Cas9 RNP and donor DNA in vivo via local administration and induce HDR. CRISPR-Nanoparticle is composed of gold nanoparticles conjugated with DNA, which are complexed with donor DNA, Cas9 RNP, and the endosomal disruptive polymer. CRISPR-Nanoparticle has shown that they deliver Cas9 RNP and donor DNA into a wide variety of cell types and in vivo by direct injection.
Each component of CRISPR system can be engineered to enhance functionality or delivery. We have identified several gRNA and donor DNA chemical modifications that enhance cell delivery of CRISPR/Cas9 therapeutics, and also enable enrichment of gene edited cells. The chemical engineering of gRNA and donor has numerous applications to enhance the delivery efficiency and tissue specificity of CRISPR. Moreover, the approach can deliver donor DNA together with CRISPR just like one molecule so that HDR efficiency can be enhanced