A breakthrough in genome engineering! The CRISPR/Cas9 (CRISPR-associated protein 9 nuclease) system uses a RNA-guided endonuclease 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.
Diagenode now offers the first antibody against CRISPR/Cas9 on the market! This antibody has been raised against Cas9 from Streptococcus pyogenes and gives excellent results in all major applications (immunoblot, immunoprecipitation and immunofluorescence). The new antibody allows for the monitoring of CRISPR/Cas9 experiments. Perform for example Chromatin IP followed by sequencing in order to make sure Cas9 is targeting your sequence of interest.
The Diagenode CRISPR/Cas9 monoclonal antibody is the first antibody able to recognize the Cas9 nuclease in a wide variaty of assays, from western blot to immunofluorescence. Moreover, with this antibody we are now able to detect not only the wild-type Cas9, but also its variants: the Cas9 nickase and fusion proteins such as the catalitically dead Cas9 fused to transcription activators VP64 and VP160, and to the transcription repressor KRAB. We will continue using this antibody to monitor the expression of these fusion proteins in different cell lines to ensure we have reached optimal levels for transcriptional modulation of target genes by the CRISPR/Cas9 system.
Patricia Renck Nunes, Prof. Lingner Group, École polytechnique fédérale de Lausanne, Switzerland
The antibody worked very well, even during our first attempt. We did a 1 hour incubation on a shaker at 4 degrees instead of overnight. We wanted to verify expression of Cas9 in various cell lines I had made (immunofluorescence). We have used the antibody a number of times and it works every time.
Researcher at Harvard University
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Better performance than tag antibodies
Figure 1: Western blot was performed on protein extracts from HEK293 cells transfected with a myc-tagged Cas9 using the Diagenode antibody against CRISPR/Cas9 as well as two commercially available purified monoclonal myc tag antibodies (supplier A and B). The three antibodies were used at different dilutions (antibody stocks: purified monoclonal Myc A 1μg/μl, Myc B 1μg/μl and Cas9 1.9 μg/μl). The ladder has been revealed by using the HRP coupled Blue ladder antibody (C15200202). Exposure time was 1 hour.
Precise localization of Cas9 by immunofluorescence
Figure 2: Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. The cells were stained with the Cas9 (A) or with an anti- Flag (B) antibody at 4°C overnight, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (bottom).
Immunoprecipitation on whole cell extracts
Figure 3: IP was performed on whole cell extracts (100 µg) from HEK293 cells transfected with a Flag-tagged Cas9 using the Diagenode antibody against Cas9. The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 shows the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 µg) are shown in lane 2 and 1, respectively.
Detection of Cas9 on protein extracts
Figure 4: Western blot was performed on protein extracts from HEK293 cells transfected with a myc-tagged Cas9 using the Diagenode antibody against Cas9. The antibody was used at different dilutions. The position of the myc-tagged Cas9 protein is indicated on the right; the marker (in kDa) is shown on the left.
Detection of Cas9 on protein extracts
Figure 5: Western blot was performed on protein extracts from HEK293 cells transfected with a myc-tagged Cas9. This figure shows the result of the WB on protein extracts from transfected (lane 2 and 4) and untransfected (lane 1 and 3) cells using the Diagenode antibody against Cas9 ( lane 1 and 2) or an anti-myc antibody (lane 3 and 4). The marker lane (M) was incubated with the Diagenode Blue ladder - HRP antibody (Cat. No. C15200202).