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Navigating Variability in Cas9 Performance Across Time

Introduction


CRISPR/Cas technology has revolutionized gene editing, providing a powerful tool across therapies, academics, and agriculture. The field is rapidly evolving, with new products and therapies constantly being rolled out. Under these high stakes, researchers are regularly challenged with the most effective reagents for their editing needs.


As a proposed solution, we have the CRISPR-Chip, a biosensor-based test system providing real-time analysis of CRISPR/Cas kinetics. This innovative platform enables scientists to interrogate gene editing activity in real-time, offering previously inaccessible data. The CRISPR-Chip can provide further data around a range of elements: gRNA activity, Cas9 performance, and the impact of environmental factors such as pH.  Various factors can impact editing outcomes; the CRISPR-Chip serves to alleviate uncertainty.


In this blog post, we delve into one specific element: storage time. We’re assessing Cas9 enzyme performance over time by comparing Cas9 activity at Day 0 versus Day 30.


Challenges in Cas Cleavage Consistency


Our lab was facing a challenge familiar to many researchers working with CRISPR: inconsistent editing outcomes. We hypothesized that storage duration of buffers could be a key contributor to variability.


Assay: A Continuation of Our Technical Note


We built upon what was outlined in our most recent technical note, "Assessing Cas9 Enzyme Performance from Different Vendors With the CRISPR-Chip Platform," further information about our assay and experiment can be found there.


The CRISPR-Chip platform, utilizing graphene field-effect transistor (gFET) technology, allows us to compare the DNA cleavage activity of Cas9 enzymes from different suppliers. Further details on the mechanics of the CRISPR-Chip can be found here.


In this specific investigation, our focus is on cleavage efficiency and understanding changes in Cas9 activity over 30 days.


Prep & Storage Conditions


We prepped the Cas9 in our buffer for assay use at 1X concentration our Dialysis Buffer contains 20mM HEPES, 100mM KCl, and 0.5mM TCEP and is pH 7.5. Immediately after the Cas9 has been prepped in our buffer, it's stored at -20C.


Methodology


We conducted a comprehensive study assessing the cleavage efficiency of Cas9 enzymes from three different vendors (anonymized as A through C) against our validated vendor. Figure A illustrates the Cas9 activity on Day 0, where Supplier C emerged as the preferred reagent, demonstrating activity most similar to our control.




Figure A showcases Cas9 activity on Day 0  

Supplier C is the preferred reagent with the highest specificity, as it has the biggest difference in millivolt response between gRNA 2 and gRNA 1.


Comparative Study of Day 0 vs. Day 30


To study the impact of storage time, we focused on the preferred reagent (Supplier C) and performed the same cleavage efficiency assay on Day 30. The results indicated a stark contrast - a lack of cleavage after 30 days. The observed decline in enzymatic activity led us to the conclusion that Supplier C would not be recommended for use at that specific time point, and confirmed our belief that elongated storage times were impacting our outcomes.



Figure B showcases Cas9 activity on Day 30

There is no significant difference in millivolt response between gRNA 2 and gRNA 1, indicating a lack of cleavage after 30 days. Due to the lack of enzymatic activity, we would not recommend any Cas9 protein to be used after 30 days of storage in the buffer, as both Cas9 enzymes tested had lost activity.



Conclusion: Navigating Variability with Interrogative Data


Our exploration regarding Cas9 variability over 30 days demonstrates the importance of quality control in Cas enzyme selection and emphasizes the importance of interrogating variables in CRISPR applications.


This study identified the most robust Cas9 cleaver (Supplier C at Day 0) and also informed us on the lack of enzymatic activity on Day 30. Beyond this, our investigation emphasizes the CRISPR-Chip's unique contribution to the gene editing landscape. As CRISPR's applications surpass the standard, adapting to its evolving capabilities and troubleshooting variability is crucial.

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