Next Generation Precision Genome Editing: Combining Multiple Advances To Create Safer Gene Therapies

However, the creation of double-strand DNA breaks (DSBs) inherent in the mechanism of wild-type CRISPR-Cas9 can lead to unintended effects. When first-generation gene editors introduce DSBs, they bring an increased risk of genomic rearrangements – especially with multiplex knockout – and the very occurrence of the DSBs can cause cell death.^1,2

To increase the precision, predictability and, thereby, the safety of genome editing technologies for use in therapeutic applications, next-generation advancements have emerged. Among these newer methods, base editing stands out because it avoids the risks that arise from the generation of DSBs.

In combination with newer engineered nucleases that can offer enhanced safety, specificity or efficiency, these two technologies together hold special promise for safer and more effective cell and gene therapies.

Base editing is precise and predictable

As a CRISPR-derived technology, base editing relies upon an RNA-guided nuclease and guide RNA specific to the genomic location that is to be edited. Instead of generating a DSB that is then repaired by the cell (usually improperly, which may result in a transcript that cannot generate a functioning protein, thus generating a knockout) as in traditional CRISPR-Cas9, base editing uses a recruited deaminase to precisely chemically alter target bases in the proximity of the bound nuclease and change one genomic letter to another (Figure 1).³

Base editing was first described with a fully deactivated Cas nuclease that retains DNA-targeting capabilities but without any cleavage activity. The enzyme itself “stands by” as the deaminase acts on the loop of single stranded DNA (ssDNA) that is created from the binding of the nuclease (called an R-loop).³ With further development, it was found that base editing efficiencies could be improved by using a ssDNA nickase version of Cas9.³

Figure 1. Traditional CRISPR-Cas9 (left) edits the genome by creating DSBs, whereas base editing (right) edits the genome by introducing predictable, precise, single base changes and avoiding DSBs. Credit: Revvity.

The clinical importance of base editing

Base editing is often thought of as a way to correct disease-causing genetic disorders caused by point mutations, where the causative mutations can be reversed through cytidine (C:G to T:A) or adenine (A:T to G:C) deamination. However, base editing is not limited to this application. Modifying just one C:G pair to a T:A pair by base editing can also disrupt splice sites or introduce premature stop codons, both of which can lead to functional protein knockout.^4,5

While correcting monogenic disorders is a critical application of base editing, using this method to knockout proteins also has significant clinical potential. For example, the generation of allogeneic (or “off-the-shelf”) cells where native surface proteins from healthy donors must be removed to “disguise” the cell therapy products from the patient’s immune system.⁶

The benefit of using base editing to make such cell therapies is in being able to achieve multiple, simultaneous edits with a reduced risk of cytotoxicity and genomic rearrangements, which are common with DSB-generating methods.

The versatile Pin-point™ base editing platform is one of the most flexible commercially available base editing systems.^6,7,8 The foundation for this platform is aptamer-based recruitment of the deaminase component through an extended guide RNA scaffold that brings together the RNA-guided nuclease and deaminase at the target locus (Figure 2). Because of the modular nature of this platform, it is amenable to the introduction of different nucleases.

When paired with a nicking nuclease, such as nCas9, the Pin-point base editing platform can accomplish high-efficiency base editing. The system can even be used to introduce multiplexed knockouts and simultaneous knock-in genotypes in a single intervention, which streamlines complex engineering projects such as those required for allogeneic CAR-T therapies.^6,7

Figure 2. The Pin-point base editing platform allows more precise genome modification by single nucleotide conversion. In one possible configuration of the Pin-point platform, shown on the left, a nickase Cas9 (nCas9) is guided to the DNA target site via a guide RNA with an aptamer engineered into the scaffold. In an alternative configuration of the Pin-point platform, shown on the right, a deactivated Type V Cas protein is guided to the DNA target site via a guide RNA with an aptamer engineered into the nuclease-appropriate scaffold. In both cases, the aptamer recruits a deaminase via fusion with an aptamer-binding protein. The combination of a nuclease, an aptamer binding protein fused to a deaminase, and an aptameric guide RNA efficiently base edit a DNA target of interest. Credit: Revvity.

Newer nucleases lay the foundation for safer therapies

While broad systematic technology advancements have given rise to base editing and other precision editing methods, continuous improvements to the nucleases used in these platforms have progressed equally quickly and offer additional promise toward safer therapies.

As a result of both the ongoing discovery of novel nucleases and further protein engineering refinement of popular Cas enzymes, the pool of powerful genome editing tools continues to grow. These newer Cas enzymes can offer greater specificity and efficiency while at the same time expanding the proportion of the genome that is accessible.

Most RNA-guided nucleases only bind to DNA that is proximal to specific recognition motifs, known as protospacer adjacent motif (PAM) sequences. These PAMs vary by type and species of nuclease. For example, the popular Cas9 nuclease, classified as Type II, recognizes a three-nucleotide NGG (and to a lesser extent NAG) PAM sequence where N is any base, accessing approximately 6% of the human genome.⁹

Type V nucleases generally recognize a four-nucleotide TTTV PAM (where V is A, C or G), which is less frequent in the human genome (only about 1%) but can provide unique access to therapeutic targets not accessible with other nucleases.⁹

Engineering new nuclease variants with relaxed PAM requirements has been an emerging area of research with recent descriptions of near PAM-less nucleases promising to significantly increase the percentage of the human genome accessible to CRISPR-based technologies.¹⁰

While accessibility is key, nucleases for next-generation therapies must also be efficient and of high fidelity. Often engineering for a balance of all these factors can result in tradeoffs of one for the other. For example, near PAM-less nucleases are not always as efficient as their PAM-requiring counterparts.¹⁰

There may also be benefits for the therapeutic use of these alternate Cas enzymes. Much of the human population expresses antibodies to Cas9, meaning exposure to a therapeutic harboring this nuclease could lead to a potentially dangerous immune response.¹¹

Combining the best methods and nucleases

To strike the best balance of maximizing both efficacy and safety for therapeutic contexts, combining new technologies like base editing with novel nucleases offers significant promise.

One Type V nuclease that offers special promise in cell and gene therapy applications is the Cas12f-derived ultra-compact CasMINI variant dCasONYX.¹² This nuclease demonstrates a superior off-target profile compared to commonly used Cas9 and Cas12a, and because it is used in a fully deactivated version for base editing, the risk of off-target DNA breaks is nearly eliminated.¹³

Additionally, this nuclease has been evaluated for its immune response activation potential and was demonstrated to have a superior immunogenicity and safety profile compared with other Cas molecules. Notably, no immunogenic responses were detected against dCasONYX across multiple human T-cell samples (unpublished data), whereas approximately 80% of the human population have prior exposure to Cas9.¹¹

Finally, the small coding length of less than 1.5 kb makes this nuclease feasible for delivery modalities such as all-in-one-vector AAV approaches, potentially enabling high-efficacy in vivo base editing treatments in the future.

The previously mentioned Pin-point platform can also be optimized to work with deactivated nucleases, including Type V CRISPR-Cas enzymes (Figure 2).⁸ The Pin-point base editing platform configured with a dCasONYX and an optimized guide RNA scaffold offer one potential configuration for a compact base editing system. Experiments using the Pin-point platform configured with dCasONYX and different guide RNAs targeting multiple genomic loci demonstrated that this configuration has robust and efficient editing capabilities.

Looking ahead

While further experiments are needed to fully evaluate the therapeutic potential of the Pin-point base editing platform configured with dCasONYX, this proof-of-concept pairing demonstrates how engineered nucleases in combination with next-generation base editing platforms are paving the way for the development of novel treatments for genetic disorders and diseases.

Advanced base editing systems like the Pin-point base editing platform have introduced an elegant way to integrate newer nucleases and deaminases. By screening different configurations of nucleases, deaminases and guide RNAs in high throughput, researchers now have a means to optimize editing of specific genomic loci.

Finding the balance between editing efficiencies, targeting capabilities, specificity and safety is a critical aspect of designing the next generation of genome editing therapies.