Overcoming Challenges in CRISPR Cas Applications
Over the past decade, CRISPR systems have revolutionized both genome editing and molecular diagnostics. This versatile technology, capable of targeting a variety of genetic sequences, has steadily progressed from enabling fundamental research through gene knockouts to powering large-scale genetic screens, ultimately culminating in the 2023 FDA approval of Casgevy, the first CRISPR-Cas9 gene therapy for sickle cell disease. Beyond Cas9, other CRISPR enzymes like Cas12a and Cas13a have proven crucial in developing rapid diagnostic tools, as demonstrated during the COVID-19 pandemic.
But to arrive at the perfect CRISPR system for a specific application, you may have to troubleshoot multiple parameters along the way. CRISPR editing requires a Cas enzyme that cuts the nucleic acid (DNA or RNA) at a specific site that is designated by a guide RNA. Additional elements include the PAM site, which has roles in Cas binding and initiating DNA unwinding. High quality Cas enzymes is an important aspect of a CRISPR experiment and for scaling up, it’s important that these enzymes produce consistent and reproducible results. Other parameters to consider are the gRNA and PAM sequence, how to best deliver the CRISPR system into your cells of interest, and the type of cut you are making (ex: Cas9 cuts dsDNA, Cas12a cuts dsDNA and RNA, and Cas13a cuts RNA).
Below, we take a look at these challenges and how to troubleshoot them for therapeutic development.
Addressing Off-Target Effects
The challenge: Off-target effects occur when Cas enzymes cut at non-target sites. This happens when the gRNA binds and directs the Cas enzyme to cut at the wrong place. Off-target effects can interfere with the function of other genes and decrease genome stability. For therapeutic applications or diagnostics, it’s important to minimize or eliminate off-target effects for patient safety and to facilitate the regulatory approval process. To characterize off-targets, scientists use whole genome sequencing or other sequencing methods to precisely map unintended genomic alterations.
Possible solutions: Scientists have been optimizing gRNAs and engineering Cas enzymes to improve their specificity. In silico prediction tools, such as CRISPRoffT can help identify potential off-target sites and help you design gRNAs that are highly specific. Engineered Cas9 variants such as SpCas9-HF1 and HypaCas9 can also minimize off-target editing.
Increasing Delivery Efficiency
The challenge: While delivering CRISPR systems in vitro has been more successful and relatively simple, delivering CRISPR systems in vivo is more complex. Delivery needs to occur specifically in a tissue of interest and must occur at high enough rates so that the CRIPSR system is delivered to all cells of interest in the region. AAVs have been ideal for therapeutic delivery because of their low immune response but they have a small packaging capacity compared to other viral vectors and may not be able to package the entire Cas gene.
Possible solutions: Alternatives to AAVs include other viral vectors such as lentiviral vectors. Although lentiviral vectors have a larger capacity, they can generate a higher immune response. Another strategy would be to use a smaller Cas enzyme such as enAsCas12f or enEbCas12a. Other alternatives include using nanoparticles such as lipid nanoparticles, gold nanoclusters, and gold nanowires for delivery. While the effectiveness of many nanoparticles have been shown in cell culture, we know less about how they work in vivo.
Avoiding Immune Responses
The challenge: CRISPR systems can generate an immune response because Streptococcus pyogenes and Staphylococcus aureus commonly infect humans. As well studied Cas9 proteins originate from these microbes, humans have preexisting adaptive immunity to Cas proteins from these organisms. An immune response to Cas proteins can cause adverse reactions and treatment failure. In addition, gRNAs can trigger the innate immune response.
Possible solutions: Using a Cas protein that originates from another bacteria that isn’t pathogenic to humans can reduce the immune response towards them. For example, a Cas12a is derived from Lachnospiraceae bacterium, a member of the human gut microbiome. If using Cas9 in clinical trials and beyond, for example, one strategy would be to detect antibodies or a T cell response towards Cas proteins (or the viral vector used) on an individual basis. To overcome immune responses towards gRNAs, phosphatase treatment of in vitro transcribed gRNAs have lower immune responses and do not affect targeting effectiveness.
Improving Enzyme Stability
The challenge: Not unique to Cas proteins, enzymes in solution can degrade over time, affecting their structure and function. Freezing enzymes can help slow their degradation but these enzymes are still subject to harsh conditions and denaturation. If you need large amounts of Cas enzyme (ex: scaling up or manufacturing), it becomes even more critical to ensure that your enzymes are stable and can produce consistent and reproducible results over time.
Possible solutions: Lyophilization extends the shelf life of enzymes at room temperature, reduces transportation costs, and increases global distribution. The extended shelf life of lyophilized Cas proteins means that you can purchase in bulk to ensure consistency and quality across all experiments or manufacturing. When paired with excipients, they can protect the enzyme from any damage during lyophilization. There have even been reports that the limit of detection of SHERLOCK, a Cas12a-based diagnostic assay, has improved with lyophilization because the test could use more sample volume.
Facilitate CRISPR Success with High-quality Cas Enzymes
Success in CRISPR applications hinges on the quality of your core components, particularly the Cas enzymes. High-quality enzymes not only ensure experimental consistency and reproducibility but also streamline the path from research to therapeutic development. For scalable applications, the ability to store enzymes through lyophilization or purchase in bulk offers additional advantages - maintaining consistency across batches, simplifying regulatory compliance, and enabling seamless scale-up. Investing in premium Cas enzymes from the start can significantly reduce experimental failures, enhance result reliability, and prove more cost-effective over time.
Fortis Life Sciences supports your CRISPR workflows with a comprehensive range of high-quality Cas enzymes, available in both lyophilized and bulk formats. Explore our selection of Cas9, Cas12, and Cas13 enzymes below.
