ACS9 Antibody

What are Cas9 antibodies and why are they important in CRISPR research?

Cas9 antibodies are immunoglobulins that specifically recognize and bind to Cas9 proteins from bacterial origins, such as Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9). These antibodies are important in CRISPR research for multiple reasons: they enable detection and quantification of Cas9 proteins in experimental systems, assist in validating genome editing efficiency, and provide insight into potential immunogenicity issues for in vivo applications. The detection of these proteins is critical for confirming successful transfection and expression in target cells, especially when troubleshooting gene editing experiments .

What is the prevalence of pre-existing anti-Cas9 antibodies in humans?

Research using validated ELISA-based assays has demonstrated that pre-existing antibodies against Cas9 proteins exist in human populations. In a study examining 200 human serum samples, approximately 10% of individuals tested positive for anti-SaCas9 antibodies, while 2.5% tested positive for anti-SpCas9 antibodies . This finding contrasts with earlier reports suggesting higher prevalence rates (79% for SaCas9 and 65% for SpCas9) based on western blotting of samples from a smaller cohort of 34 donors . These antibodies likely result from natural exposure to these common human pathogens, as 58% of the donors tested positive for Group A Streptococcus in the same study .

Why does the prevalence of anti-SaCas9 antibodies differ from anti-SpCas9 antibodies?

The difference in prevalence (10% for anti-SaCas9 versus 2.5% for anti-SpCas9) likely reflects the different colonization rates of the source bacteria in humans. S. aureus has a reported colonization rate of 26.2% in humans, while S. pyogenes has a lower rate of 9.6% . Additionally, the immunogenicity of the proteins themselves may differ, with SaCas9 potentially presenting more immunogenic epitopes than SpCas9. This distinction is important for researchers selecting a Cas9 variant for in vivo applications, as SpCas9 might trigger fewer immune responses in human subjects .

How can ELISA-based assays be optimized for detecting anti-Cas9 antibodies in human sera?

Optimization of ELISA-based assays for anti-Cas9 antibody detection requires careful consideration of multiple parameters. First, researchers should determine the minimum required serum dilution that maintains at least 80% of the dynamic range of the assay. Studies have shown that a 1:20 dilution is optimal, providing sensitivity of 2.93 ng/mL for anti-SaCas9 and 3.90 ng/mL for anti-SpCas9 antibodies .

For detection, horseradish peroxidase (HRP)-coupled protein G can be used to detect antibodies binding to both SaCas9 and SpCas9. The assays should be standardized using appropriate controls, such as rabbit polyclonal anti-SaCas9 antibody and mouse monoclonal anti-SpCas9 antibody. The dynamic range for anti-SaCas9 antibody detection is typically 0.73–750 ng/mL, while anti-SpCas9 antibody detection ranges from 0.24–1,000 ng/mL . Researchers should also validate assay specificity by confirming that anti-SaCas9 antibodies do not cross-react with SpCas9 and vice versa.

What considerations are important when establishing cut points for screening and confirmatory assays for anti-Cas9 antibodies?

Establishing appropriate cut points for anti-Cas9 antibody assays is critical for accurate identification of positive samples. Two methods are commonly employed:

• Traditional method: Using untreated serum samples from a training set (typically 48 healthy donors), assuming a false-positive rate of 5%. This method yielded screening cut points of 1.012 (OD 450) for anti-SaCas9 and 0.874 (OD 450) for anti-SpCas9 antibodies .

• Immune-inhibition method: Pre-treating samples with excess Cas9 (200 μg/mL) prior to establishing cut points. This approach resulted in lower screening cut points: 0.5129 (OD 450) for anti-SaCas9 and 0.6146 (OD 450) for anti-SpCas9 antibodies .

For confirmatory assays, competitive inhibition tests determine the specificity of positive samples identified in screening. Diluted serum samples (1:20) are incubated with or without excess free Cas9 (200 μg/mL) to calculate percent inhibition. The confirmatory cut points were determined to be 71.61% inhibition for anti-SaCas9 and 73.11% inhibition for anti-SpCas9 antibodies . Statistical methods involve removing outliers outside 1.5 times the interquartile range and using mean percentage inhibition plus 1.645 times the standard deviation as the cut point for signal inhibition.

How can IgY polyclonal antibodies against SpCas9 be produced for research applications?

Production of IgY polyclonal antibodies against SpCas9 can be achieved through a one-month immunization scheme in hens. This method offers advantages over mammalian antibody production, including non-invasive collection and reduced cross-reactivity with mammalian systems. The process involves:

• Immunizing hens with purified SpCas9 protein

• Collecting eggs after sufficient immunization period

• Isolating antibodies by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively

This approach yields highly sensitive and specific antibodies that can detect SpCas9 in various biological samples, including parasitic protozoa such as Leishmania braziliensis . The resulting anti-SpCas9 IgY polyclonal antibodies can be used at dilutions of 1:1000 (from eggs) or 1:5000 (from immune blood) as primary antibodies in western blot applications, with anti-IgY (1:10,000) coupled with alkaline phosphatase as the secondary antibody .

How can CRISPR-Cas9 be used to validate antibody specificity?

CRISPR-Cas9 technology offers a robust approach for validating antibody specificity by creating knockout cell models that serve as negative controls. The process involves:

• Designing single guide RNA (sgRNA) molecules that target the gene encoding the protein of interest

• Introducing the sgRNA along with Cas9 endonuclease into cells

• Allowing Cas9 to cleave the target DNA, resulting in gene knockout

• Using the resulting knockout cells as negative controls for antibody validation

This approach provides a powerful method to verify that an antibody recognizes its specific target, as the antibody should not produce a signal in the knockout cells if it is truly specific. CRISPR-Cas9 also enables simultaneous testing of antibody specificity for multiple signaling proteins in a pathway by knocking out expression of upstream mediators, allowing for streamlined and high-throughput validation .

What are the implications of pre-existing anti-Cas9 antibodies for in vivo CRISPR-based therapies?

Pre-existing anti-Cas9 antibodies have several potential implications for in vivo CRISPR-based therapies:

Therefore, assessing immunogenicity of all CRISPR/Cas9-based therapeutic products is recommended, with risk assessment addressing whether the therapeutic elicits anti-drug antibodies and what clinical consequences these antibodies might have .

How can anti-Cas9 antibodies be used to detect Cas9 protein in biological samples?

Anti-Cas9 antibodies can be effectively used to detect Cas9 protein in various biological samples through several techniques:

• Western Blotting: Total protein extracts are prepared from biological samples (e.g., 10 mL of exponential phase parasites collected by centrifugation), subjected to SDS-PAGE, and transferred to PVDF membranes. Anti-SpCas9 IgY antibodies (1:1000 from eggs or 1:5000 from immune blood) can be used as primary antibodies, with anti-IgY (1:10,000) coupled with alkaline phosphatase as the secondary antibody .

• ELISA: Direct format ELISAs can detect Cas9 proteins with high sensitivity. For SpCas9, the assay has a dynamic range of 0.24–1,000 ng/mL and a sensitivity of 0.24 ng/mL. For SaCas9, the assay has a dynamic range of 0.73–750 ng/mL and a sensitivity of 0.73 ng/mL .

• Immunofluorescence: Anti-Cas9 antibodies can be used to visualize the subcellular localization of Cas9 proteins in fixed cells, providing insight into the distribution and expression levels of the protein.

These methods are particularly valuable for confirming successful transfection and expression of Cas9 in experimental systems, especially in models where genetic modification efficiency may be variable or challenging to assess by other means.

Prevalence of Pre-existing Anti-Cas9 Antibodies in Human Sera

Note: Data from study of 200 human serum samples using both traditional and immune-inhibition methods for cut point determination .

Sensitivity and Dynamic Range of Anti-Cas9 Antibody Assays

Note: Data from ELISA-based assays developed for detection of anti-Cas9 antibodies .