RGENs- CRISPRs

CRISPR technology

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein (Cas) systems are adaptive mechanisms evolved by bacteria and archaea to repel invading viruses and plasmids. Recently, efficient genome editing by the CRISPR-Cas system has been shown in multiple organisms, including zebrafish, mice, rats, C. elegans, plants, and bacteria. Several groups have demonstrated that compared with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Cas–mediated gene targeting has similar or greater efficiency in cells and zebrafish.

CRISPR nucleases, also called RNA-Guided ENdonucleases (RGENs) are therefore, programmable genome engineering tools where the Cas9 forms a sequence-specific endonuclease when complexed with two RNAs, one of which guides target selection. Likewise, RGENs consist of constant components (Cas9 and tracrRNA) and a target-specific RNA (crRNA).

We then developped several applications based on the CRISPR/cas9 system that are available now as DNA vector based systems (dRGENs) or injectable RGENs (aRGENs):

- Gene knockout

- Gene Knock-In, gene tagging or Gene mutagenesis

- Safe Harbour gene integration (AAVS1 for human or rosa locus for mouse)

- Gene knock-down: CRISPRi (CRISPR interference)

- Gene activation: CRISPR-TF allow specific artificial gene activation

Also available from LabOmics are Cas9 mRNA, Cas9 recombinant proteins (wild type and nickase) and Cas9 expression vectors (including lentiviral vectors)

DNA-directed RGENs (dRGENs):

Design and synthesis of plasmids vectors encoding for the CRISPRs targeting your sequence, gene or genomic region of interest. The single-guide RNA (sgRNA),consisting of crRNA and tracrRNA, and the Cas9 protein can be expressed from 2 different plasmids to be co-transfected or from one plasmid expressing the whole system. Selection markers are also available for our CRISPR vectors, such as hygromycin, neomycin, puromycin with or without co-expression of mCherry or eGFP.

Multiple sgRNA clones can be constructed and co-transfected with one Cas9 clone to enable simultaneous editing of several sites within the genome, offering greater efficiency and flexibility for the experiment design.

Different Cas9 expression vectors are available too:

- Cas9 wild type in mammalian expression vector or lentiviral vector, co-expression of eGFP or not.

- Cas9 D10A "nickase" in mammalian expression vector, co-expression of neoR and mCherry or not.

Active RGENs (aRGENs):

All components of RGENs will be in active form (RNA components as in-vitro-synthesized RNAs, Protein component in purified recombinant protein). When mixed they are active enzyme. It is a perfect option for researchers who are planning to make KO animal by direct cell injection (c.elegans, drosophila, zebrafish, medaka, mouse, rat, rabbit and etc.). aRGENs also can be directly delivered into cultured cells (by electroporation / possibly by protein transfection reagents) to induce efficient genome engineering.

Deliverables:

- RNA: crRNA, tracrRNA or sgRNA

- Cas9 wild type mRNA or CAS9 wildtype recombinant active protein

- Cas9 D10A "nickase recombinant active protein