"CRISPR", or clustered regularly interspaced short palindromic repeats, is an ancient bacterial mechanism that prevents the invasion of foreign pathogens to a host organism. Specifically, the CRISPR sequence has been identified as a single DNA sequence that is repeated with unique sequences (found to be that of viruses) in-between. Thus, bacteria have created an environment that allows them to recognize and attack viruses in case of a re-invasion. "Cas", or CRISPR-associate protein, is the second part of this defense mechanism, and is responsible for cutting targeted DNA and viruses. Cas9 is the best-known "Cas" enzyme and originates from Steptococcus pyogenes (or strep throat). Together, CRISPR/Cas9 work to fill the CRISPR region with foreign DNA and turn on Cas9's splicing machinery. Next, the copied viral RNA and Cas enzymes identify viral material and slice it apart to avoid further replication. Since the discovery of this ancient defense mechanism's potential in gene editing and disease therapies, the CRISPR/Cas9 primary antibody has been helpful in determining new avenues of research.
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Immunocytochemistry/Immunofluorescence: Cas9 Antibody (7A9-3A3) [NBP2-36440] - Hela cells were transiently transfected with an N-terminally Flag-tagged S. pyogenes Cas9 expression vector. The cells were stained with the Cas9 antibody followed by anti mouse-AF488 coupled secondary antibody. Nuclei were counter-stained with Hoechst 33342.
Chu et al used a CRISPR/Cas9 antibody to test the generation of Rosa26 knock-in alleles for the insertion of transgenes in order to study homology-directed mutagenesis in inbred embryos. Rosa26 is a locus that is often used for the integration of transgene constructs to reach altered gene expression in mice. The traditional method of targeting genes in this model requires specific ES clones to be injected into blastocysts so that these alleles are transferred their offspring. However, this procedure is timely and costly, therefore CRISPR/Cas9 was introduced into these embryos instead. A CRISPR/Cas9 antibody was used in western blot to determine whether the Rosa26-LSL-Cas9 generated allele was in fact functional. This expression was analyzed in western blot using the CRISPR/Cas9 antibody against lysates of Tat-Cre treated B cells, where the expression was verified. Overall, the use of a CRISPR/Cas9 antibody helped to establish the direct targeting of experimental compound mutants without the cost and time normally associated.
Chiou et al also used a CRISPR/Cas9 antibody in their creation of a Cre-regulated Cas9 allele that allows for the retrograde study of pancreatic ductal adenocarcinoma (PDAC). Similar to Chu et al, developing genetically engineered mouse models of PDAC is extremely laborious and costly, and the introduction of the CRISPR/Cas9 system allows for fast takeovers of gene function during the metastasis of this tumor type. A CRISPR/Cas9 antibody was used in western blot to validate the generation of cre-regulated Cas9 mice in fibroblasts. This group successfully developed a method to alter PDAC tumor development on a molecular level, for any gene of interest, without the creation of a new mouse model. Overall, in the early stages of CRISPR/Cas9 gene editing discovery, CRISPR/Cas9 antibodies have proven to be critical to the development of new applications.
PMID: 26772810
PMID: 26178787
PMID: 27398291
