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CRISPR/Cas9 Proteins

CRISPR-associated (Cas) Proteins for Genome Editing

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in select bacteria like Streptococcus pyogenes. Cas9 induces site-directed double strand breaks in DNA leading to gene inactivation or the introduction of heterologous DNA sequences through non-homologous end joining (NHEJ) and homologous recombination (HR).

Transfecting Cas9 protein/gRNA ribonucleoprotein (RNP) complexes versus the use of plasmid-driven expression systems offers high editing efficiency, rapid cellular response, transient activity and lower off-target effects.

Products

Cas9 - wide-type protein. Endonuclease activity resulting in a double-strand cut.

Applications: useful for testing guide RNA in vitro or for site-specific cutting of DNA vectors or in vitro DNA

Cas9 (biotin) - biotinylated wide-type protein. Retains full endonuclease activity.

Applications: useful for assays surface binding of active Cas9. Cellular Cas9 availability studies.

Cas9 (fluorescent conjugate) - fluorescent wide-type protein. Retains full endonuclease activity.

Applications: useful for cellular Cas9 availability studies.

Cas9-NLS - Cas9 with nuclear localization sequence on the C terminus. for efficient entry of the Cas9 protein into the nucleus.

Applications: useful for cell editing experiments and for microinjection of animal embryos for creating transgenic animals (knock-out or knock-in)

Cas9 D10A - amino acid change at position 10 in the Cas9 amino acid sequence inactivating the nuclease catalytic activity. Produces a nick (single-strand cut) rather than a double-strand cut in DNA.

Applications: improves editing specificity at the target site by requiring the creation of two guide RNAs to target each DNA strand around the target site to make the double-strand cut

Cas9 H840A - amino acid change at position 840 in the Cas9 amino acid sequence inactivating the nuclease catalytic activity. Produces a nick (single-strand cut) rather than a double-strand cut in DNA.

Applications: improves editing specificity at the target site by requiring the creation of two guide RNAs to target each DNA strand around the target site to make the double-strand cut

Cas9 D10A/H840A - endonuclease dead Cas9 (dCas9); still retains guide RNA-directed DNA binding activity.

Applications: used for temporary interference of gene expression and DNA binding assays