Cas9 Antibodies: Overview & Applications

Cas9 Antibodies: Overview & Applications

Cas9 Antibodies: Overview

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas is one of the more advanced and widely used tools for genome editing. The system was originally discovered in E. coli genomic sequences in 1987. However, the components and working mechanism of the CRISPR/Cas system were fully understood in 2011. Adding the final piece of the puzzle, trcrRNA (or trans-activating CRISPR RNA), in vitro by Emmanuelle Charpentier’s group opened the door to gene editing applications. 

The CRISPR-Cas9 system is composed of the following components:

  • A guide RNA (or gRNA): About 20 bases long and involved in matching the target gene. The scaffold part of the gRNA binds to the target DNA and the rest sequence is involved in guiding Cas9 endonuclease to the target site to perform double-stranded DNA cleavage. 
  • Cas9 (CRISPR-associated protein 9): An endonuclease enzyme of 160 kDa and acts as a molecular scissor. It’s involved in cleavage and modification of the target genome. Though variant forms of Cas9 proteins are available today, the most commonly used nuclease enzyme is isolated from Staphylococcus aureus (SaCas9) and Streptococcus pyogenes (SpCas9).

Currently, researchers are working on modifying these components to expand the applications of the system in biomedical research and effective treatment of critical diseases, such as cancer and genetic disorders. 

However, the major limitation associated with the application of CRISPR-Cas9 in biomedical applications is its immunogenicity due to Cas9, ineffective delivery system, and ethical concerns. 

Therefore, different immunological assays are used in the therapy development and application to assess the level of Cas9 protein in a biological sample. By doing so, researchers can understand the potential immunogenicity in patients and develop effective strategies for the concerned disease treatment. 

In this article, we will review how Cas9 antibodies work, their functions and applications in labs and industries, what options of commercially available Cas9 antibodies are available, and the applications that require the reagent. 

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How Do Cas9 Antibodies Work?

CRISPR/Cas9 system was first found as an adaptive immune system in microbes. The RNA-guided DNA endonucleases are involved in the cleavage of the foreign DNA molecule with the help of guide RNA, which is composed of crsprRNA and trcrRNA. The system is frequently used in gene therapy and gene editing applications. 

However, gene therapy involving the CRISPR-Cas9 system has been observed to cause immunogenicity in patients. For example, some studies have identified T cells’ reactivity to Cas9 antigens in donors, suggesting the presence of a pre-existing immune response. That’s why Cas9 antibodies are developed to monitor the enzyme level in patients. 

A range of commercially available Cas9 antibodies for diverse applications include recombinant rabbit monoclonal antibodies, rabbit polyclonal antibodies, and Cas9 monoclonal antibodies. They are used in immunoassays that generally involve both primary antibody and secondary antibody (with HRP or AP conjugates), such as ELISA, Immunofluorescence, IP, IHC, Chromatin Immunoprecipitation (ChIP), ICC, IHC-Paraffin, Simple Western, Flow Cytometry, and Western Blot.

It’s recommended to go through the safety data sheet (SDS), images, reviews, citations, and other technical details provided by antibody distributors for the validation of the efficacy and usefulness of respective Cas9 antibodies in your experiment. 

Cas9 Antibodies

A variety of Cas9 antibodies have been developed for various applications. For example, a 7A9 mouse monoclonal antibody, supplied by Epigentek, is developed using a synthesized peptide that corresponds to the sequence of the Streptococcus pyogenes (S. pyogenes) CRISPR-associated endonuclease Cas9/Csn1. 

This Anti-Cas9 monoclonal antibody has the capability to identify the presence of CRISPR/Cas9 expression in target cells using a range of techniques such as Western blotting (WB), immunofluorescence (IF), immunoprecipitation (IP), or enzyme-linked immunosorbent assay (ELISA). The use of this test lies in the confirmation of successful transfection of gRNA and Cas9 vectors.

Similarly, the Guide-it Cas9 Monoclonal Antibody (Clone TG8C1), supplied by TakaraBio, is a mouse-derived antibody created from hybridoma cells targeting the full-length recombinant Cas9 protein sourced from Streptococcus pyogenes. 

This antibody can detect wild-type Cas9 in mammalian and bacterial cell lysates at concentrations of less than 1 ng when utilized in Western blot assays.

Rabbit-derived antibodies, Guide-it Cas9 Polyclonal Antibody, are also available that are developed against recombinant Cas9 protein from Streptococcus pyogenes. It targets three Cas9 nuclease variants with high specificity and sensitivity, such as wild-type Cas9, Cas9 nuclease-deficient mutants, and the Cas9 nickase.

What are Cas9 Antibodies Used For?

CRISPR-Cas9 is a widely recognized and used tool in biotech and biomedical labs for faster and cheaper genome editing applications with high sensitivity, such as

  • Cas9 protein detection using Western blots
  • Cas9 protein detection by immunocytochemistry

Western Blots

Western blotting is routinely used in labs and industries to detect and quantify proteins. It works on the principle of antigen-antibody interaction. It utilizes Cas9 antibodies to validate Cas9 expression in cell lysates and assess the efficiency of transfection methods. 


Immunohistochemistry is a method for detecting and localizing specific antigens in cells and tissues by exploiting specific antibody-antigen interactions. It utilizes Cas9 antibodies to localize Cas9 protein within tissues or cell samples and assess the safety of CRISPR-Cas9-based therapies.  

What Industries Use Cas9 Antibodies?

Cas9 antibodies have extensive applications in the Biotech and Biomedical industries for research and diagnostic purposes. 


In biotech fields, Cas9 antibodies are used to assess the expression level of Cas9 proteins in different cell and tissue samples. It’s used in a range of assays, such as Immunoprecipitation, Immunocytochemistry, Western Blot, ELISA, and Immunohistochemistry. for the analysis of Cas9 proteins in Human and Mouse samples.


CRISPR-Cas9 system's role in bacterial immune response has opened up a world of new possibilities for treating a wide range of diseases in humans. It’s utilized in both ex vivo and in vivo editing applications. 

However, using this technology in vivo triggers an immune response, which is a major limitation of the technology’s application. Consequently, the presence of pre-existing antibodies against Cas9 could be a significant consideration during clinical development.

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Cas9 is an RNA-guided DNA endonuclease enzyme involved in the cleavage of target DNA fragments in organisms. It works in association with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to provide adaptive immunity to microbes against viruses.

The CRISPR/Cas system is widely used in gene editing applications in biotech and biomedical fields. However, given the immunogenicity observed in some patients receiving CRISPR-Cas9-based therapies, the Cas9 antibody becomes an essential tool for monitoring Cas9 protein levels across various samples. It plays a pivotal role in assessing the degree of immunogenicity that may occur in patients undergoing therapy, enabling the development of more effective treatment strategies for diseases. 

The successful execution of CRISPR-Cas9-based applications demands the use of advanced equipment and high-quality reagents to ensure the precision and consistency of results. This approach minimizes the need for repeated experiments due to data variability within any given experimental set.

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