CRISPR/Cas9

Genome Engineering Tools from Novabiotein

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes. S. Pyogenes utilizes Cas9 to remember and later probe and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. When the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. This ability to make site-directed double strand breaks in DNA has led to the use of Cas9 protein as a genome engineering tool. As an RNA-guided DNA endonuclease, Cas9 can be directed to specific target cleavage sites by altering the guide RNA sequence. The nuclease-deactivated form of dCas9 further provides a versatile tool for biomedical research and therapeutics.

Stratech Scientific offers a comprehensive range of CRISPR/Cas9 related enzymes, proteins, antibodies and kits for genome engineering from Novateinbio.

Transfection-ready Cas9 proteins

Compared to transfection with Cas9-expressing plasmid, delivery of purified Cas9 protein directly into cells by electroporation is relatively straightforward and may reduce the risk of issues associated with plasmid-based transfection, such as random integration of plasmid into the host genome. The presence of a nuclear localization signal (NLS) localizes the Cas9 RNP complex to the nucleus immediately upon entering the cell with the need for transcription or translation compared with mRNA or plasmid systems. Additionally, the Cas9 RNP complex is rapidly cleared from the cells minimizing the chance of off-target cleavage when compared to other systems. There is no need for transcription or translation processes.

NLS-Cas9-NLS

Cas9 NLS (PR-137211), is a recombinant dCas9 from S. pyogenes which contains Simian virus 40(SV40) T antigen nuclear localization sequence (NLS) on the N- and C- termini of the protein and is used to create double blunt end for recombination. The protein is transfection-ready for gene editing, also used for sgRNA or crRNA screening.

NLS-dCas9-NLS

NLS-dCas9-NLS (PR-137213B) is an engineered, nuclease- deactivated Cas9 (dCas9) protein, has been used to regulate endogenous gene expression and labelling of genomic loci in living and fixed cells. The protein has been mutated CRISPR-associated endonuclease Cas9 (amino acids 1 to 1368) with D10A & H840A (ACCESSION: AKS40378 for Cas9). To facilitate nuclear entry, two nuclear localization signal sequence (NLS) are fused to both N- and C-terminal of dCas9 protein.

NLS-Cas9-EGFP

for subcellular localization of the Cas9-gRNA/target DNA complex.
NLS-Cas9-EGFP (PR-137211E) is a fusion protein containing a nuclear localization sequence (NLS) on its N terminal and EGFP on the C terminal. The EGFP can be taken as a reporter for tracking or sorting transfected cells, to enrich for cell populations containing the desired genome edits by fluorescence activated cell sorting (FACS). It significantly reduces the labour and cost associated with single cell cloning and genotyping in genome editing applications.

NLS-Cas9(D10A)-2NLS nickase

NLS-Cas9(D10A)-2NLS nickase (PR-137212B) is a transfection-ready, recombinant Cas9 for gene editing, single nick creation on double strand DNA. NLS-Cas9-D10A Nickase is a mutation form of Cas9 Nuclease which makes one active domain deactivated, thus it can only cut one single strand DNA that is complementary to the guide- RNA, producing one single strand cut. Combined with two different gRNA, NLS-Cas9-D10A Nickase produces two cut sites respectively and causes a double strand break. Compared with the wild type Cas9 nuclease, the two- gRNA guided cleavage can significantly reduce the off-target effects.

Screening Tools for CRISPR/Cas9