Application

Monoclonal Anti-CRISPR/CAS9 (C-terminal) recognizes CAS9 protein in CAS9 construct over-expression systems. The antibody successfully recognizes mutant Cas9 variants, including nickase Cas9 and dead Cas9 (dCas9). The antibody may be used in various immunochemical techniques including Immunoblotting, Immunofluorescence and Immunoprecipitation. Monoclonal Anti- CRISPR/CAS9 (C-terminal) does not cross react with FnCas9 from Francisella novicida bacteria and Cpf1 proteins from Acidaminococcus sp. (strain BV3L6) and Lachnospiraceae bacterium ND2006.

Biochem/physiol Actions

CAS9 plays a vital role in plasmid DNA interference. It is the only Cas protein needed to deliver resistance against foreign DNA. CAS9 stimulates both RNA-guided genome editing and gene regulation in various organisms, but it can facilitate only one activity at a time within any given cell.

General description

CAS9, also known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease, is the signature gene of the type II CRISPR -RuvC (RNase H-like fold) Cas system. CAS9 contains 1388 amino acids. This protein is predicted to contain a RuvC/ ribonuclease (RNase) H domain involved in CRISP RNA (crRNA) maturation and McrA/HNH signature domain involved in the DNA degradation step.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) belongs to the type II CRISPR/CAS9 system. It is part of an adaptive immune system of the Streptococcus pyogenes SF370, protecting from pathogens′ target genes by cleaving the foreign DNA in a sequence-dependent manner. The type II CRISPR/Cas system which has been adapted to expression in eukaryotic cells, consists of four genes including the Cas9 (CRISPR-associated proteins) nuclease, two noncoding CRISPR RNAs (crRNAs, or gRNA), trans-activating crRNA (tracrRNA) and a precursor crRNA (pre-crRNA) array. The pre-crRNA contains nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs). The Cas9 endonuclease can be engineered with a single gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB. In comparison to other genome-editing technologies such as designer zinc fingers (ZFs), transcription activator–like effectors (TALEs) and homing meganucleases, the CRISPR/CAS9 system is a scalable, affordable and easy to engineer. Therefore, the anti-CRISPR/CAS9 antibody can be a useful tool for detecting CRISPR/CAS9 positively transfected cells, revealing DSB sites in the genome and in ChIP (Chromatin Immunoprecipitation) related assays

Immunogen

Recombinant protein within the C-terminal region of Streptococcus pyogenes Cas9

Other Notes

This product is for R&D use only, not for drug, household, or other uses. In order to obtain best results in different techniques and preparations we recommend determining optimal working concentration by titration test.

Physical form

Supplied as a solution in 0.01 M phosphate buffered saline pH 7.4, containing 15 mM sodium azide as a preservative.

Storage and Stability

For continuous use, store at 2-8 °C for up to one month. For extended storage, freeze in working aliquots. Repeated freezing and thawing is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use. Working dilution samples should be discarded if not used within 12 hours.