Biomedical researchers have long sought the “magic wand” for targeted genome editing in an attempt to affect phenotypic outcome. A number of methods allow manipulation of gene function, including viral vectors, homologous recombination, RNAi, and nuclease-based gene editing systems.These approaches, however, are costly, time-consuming, and unsuitable for large-scale studies.

The most recent CRISPR/CAS9 gene editing technology uses a naturally occurring bacterial nuclease from Streptococcus pyogenes (Sp). The Cas9 system uses a RNA-guidedendonuclease technology which allows for inducing indel mutations, specific sequence replacements or insertions and large deletions or genomic rearrangements at any desired location in the genome. In addition, Cas9 can also be used to mediate up- or downregulation of specific endogenous genes or to alter histone modifications or DNA methylation. In this system, a single multi-domain Cas9catalyzes a double-strand break in the target DNA composed of a 20-bp sequence matching a guide RNA (gRNA) protospacer and anadjacent downstream 5’-NGG nucleotide sequence (the protospacer-adjacent motif (PAM)) that can be targeted to precise genomic locations. Since the Cas9 nuclease is targeted to its cognate DNA locus through a simple RNA sequence, CRISPR/Cas9 is more powerful and versatile than other system that uses protein- based targeting, requiring only RNA redesign to change target specificity.


Three main applications of the S. pyogenes Cas9 nuclease have been described, each which relies on a specific Cas9 protein. The first relies on the use of a nuclear localized Cas9 protein bearing wild-type activity to induce single double strand break leading either to NHEJ (Non-Homologous End-Joining) or to HDR (Homology Directed Repair). Without a homologous repair template, NHEJ can result in indel mutations, disrupting the target sequence. Alternatively, targeted mutations can be made with a homologous repair template and by exploiting the HDR pathway. The second, a mutated Cas9 protein, Cas9D10A, exhibits only site-specific single-strand nicks and does not activate NHEJ, enabling extra precision in gene targeting. Using a pair of sgRNA-directed Cas9 nucleases, it is possible to induce large deletions or genomic rearrangements, such as inversions or translocations. The third protein, nuclease-deficient Cas9 or dead Cas9 (dCas9), two single point mutations inactivate Cas9 cleavage activity, but do not alter DNA binding to its cognate DNA target locus. This dCas9 can be used to targetvarious effectors (i.e. transcriptional activators, repressors, fluorescent proteins) in a sequence- specific manner without cleavage. This dCas9can mediate the up- or downregulation of specific endogenous genes or alter histone modifications or DNA methylation at any genomic location. Cas9 from other species than S. pyogenes have their own PAM sequence and interesting properties. Cas9 from S. aureus (SaCas9) has similar gene editing efficiency to that of SpCas9, but is 1kb smaller. This is ideal for adeno-associated virus (AAV) delivery, which has a packaging limitation of ~4.5kb. Recently, a newly characterized class II nuclease called Cpf1 promises to deliver simple and precise genome engineering by relying on cutting properties, compactness and ability to rely on just one RNA. Cpf1 interesting features portend a future for genomics in which researchers will have any number of editing tools depending on the material being edited.


A good CRISPR-Cas9 antibody is more than just specific. Attempts to generate codonoptimized Cas9 to increase Cas9 expression efficiency have not been successful. Therefore it is critical to choose highly validated antiCas9 antibodies for downstream applications. A number of methods now also require more than a simple check of the expression of Cas9using an untagged antibody. Diagenode has developed the largest and most comprehensive set of anti-SpCas9 antibodies, raised against the N or C-terminus of Cas9 nuclease, to address a range of research needs. Each antibody has been highly validated for very specific applications: •Chromatin immunoprecipitation (CRISPR/ Cas9 polyclonal antibody) •Immunofluorescence, western blot, and immunoprecipitation (extremely specific clone 4G10; clone 7A9) •Experiments with fusion proteins – using an antibody raised against C-terminus of Cas9protein, validated in IF, WB and IP.


CRISPR-Cas9 technology is a powerful tool for cell genome reprogramming and molecular biology research. This genome-editing system gives remarkable new possibilities due to its simplicity, high efficiency, and versatility. High quality anti-Cas9 antibodies are crucial for the detection of the Cas9 protein in the edited experimental system.