The discovery of CRISPR-Cas gene editing technology has revolutionized biological research, providing new insights and opportunities for the advancement of several industries. However, the technology’s future depends on the identification and development of novel Cas nucleases.
Certain properties found in novel Cas proteins present advantages for different applications. Some nucleases exhibit expanded targeting capabilities thanks to their ability to recognize distinct protospacer adjacent motif (PAM) sequences, whereas others can be applied using various delivery methods due to diversity of nuclease sizes. Moreover, Cas nucleases exhibit diverse DNA-binding affinities, enzymatic activities, and thermostabilities, allowing for application-specific tailoring.
Novel Cas families, naturally, find their niches in emerging applications and industries. One example is the discovery and development of novel Type V and Type VI Cas nucleases with collateral activity. Such nucleases have been critically important in the advancement of diagnostics dependent on nucleic acid detection.
Novel Cas nucleases also present opportunities to create new types of gene editing. For example, catalytically inactive Cas nucleases enable base, prime, and epigenome editing. And Cas nickases avoid the introduction of double-stranded DNA breaks. Novel Cas nucleases have expanded research possibilities and addressed major CRISPR limitations, such as off-target effects.
Moreover, the characterization of these novel Cas nucleases has opened new avenues for precise genome manipulation, allowing researchers to address specific gene editing challenges across various industries. From agriculture to medicine, the diverse characteristics of Cas proteins have created opportunities for innovative applications, driving the growth and diversification of CRISPR-Cas technology even further.
But how are novel Cas nucleases identified, classified, and applied in practice?
Initial steps
Caszyme has built expertise in identifying unique Cas proteins. Company scientists recently identified and characterized a new family of Type V CRISPR nucleases from a relatively unexplored phylum of bacteria, Armatimonadota. It was done by searching microbial sequence datasets for CRISPR-associated nucleases that contained a single RuvC domain encoded in an operon-like organization with cas1 and cas2 genes.
New systems were defined by studying their locus gene architectures, by inspecting the putative effector nuclease structures, and by carrying out phylogenetic analyses. Next, each system’s ability to hydrolyze double-stranded DNA was evaluated by expressing the candidate CRISPR loci in Escherichia coli and using the subsequent lysates to treat a plasmid DNA library with randomized PAM sequences. Each system’s PAM recognition was determined by complexing it with its complementary single-guide RNA (sgRNA) and then performing incubation and sequencing. Results were confirmed via interrogation using substrates with fixed nonrandomized PAM sequences.
The identity of the sgRNA responsible for directing the systems also had to be determined. For that, a 12–13 bp region with complementation to the CRISPR repeat—an anti-repeat—was found by searching the noncoding region between the nuclease and Cas genes. Sequence analysis of this region showed that it was likely to form RNA secondary structure motifs, a feature observed in the sgRNA of some other Cas systems. This information was then used to design and manufacture system-specific sgRNA sequences.
Next, system cleavage efficiency was assessed using 12 targets from two therapeutically relevant human genes, WRAP and RunXI, at 37°C. The effects of DNA supercoiling and varied temperatures up to 50°C were also observed. Finally, the Cas systems were assessed for whether they induced degradation of single-stranded DNA after DNA target recognition. This was done by incubating the systems in the presence or absence of single-stranded DNA or double-stranded DNA target and single-stranded DNA from the bacteriophage M13, with degradation of bacteriophage single-stranded DNA being the observed metric.
Applications in human biology
Initial characterization of wild-type Cas systems in many cases reveals limited activity in human cells, but since a large proportion of CRISPR research is focused on developing human-oriented therapeutics, the next step for researchers is to engineer these systems by increasing their activity in human cell lines. Through a combination of various rational protein and RNA design approaches, guided by structural data, Caszyme’s scientists have been able to refine the biochemical properties of Cas systems, increasing their efficiency, thus widening their suitability for various gene editing applications.
Although this tutorial will not offer many specifics of this process (after all, this is the “secret sauce” of our work), one example we can highlight is that of the Cas12l family which, post-engineering, demonstrated significantly increased gene editing tool activity in eukaryotic cells up to 10-fold on some targets (Figure 1).
Cas12l belongs to a distinct protein family that has no sequence homology to other Cas12 nucleases, making it difficult to accurately predict its structures. Cryo-electronic microscopy provided the solution. Structural analysis with this process revealed moderate structural resemblances between Cas12l and just a few other families of Cas12 enzymes. It also highlighted key architectural features essential for effector complex assembly. Furthermore, distinct structural motifs were identified that “lock” the DNA substrate within the ternary complex poised for cleavage. Leveraging this structural insight, Caszyme fine-tuned its engineering of Cas12l, resulting ultimately in an increase of its activity in human cells.
The road ahead
Although progress has been made in both the discovery and engineering of Cas nucleases, the journey toward clinical translation of CRISPR-Cas technology still presents several challenges, one of them being delivery. The efficient delivery of CRISPR components—including Cas nucleases, sgRNAs, and donor DNA templates—is crucial for achieving precise, targeted gene editing in vivo. Other important tasks include managing the interplay between delivery methods, targeting different cell types, and overcoming naturally occurring in vivo barriers. With each delivery method offering unique advantages and challenges, it is important to remember that optimization of delivery compositions is not just a technical aspect that needs to be taken into consideration somewhere along the way, but it is also a crucial step toward ensuring the safety, efficacy, and scalability of CRISPR-based therapies.
As companies and academic institutions work on overcoming these challenges, great future promise emerges in precision medicine, diagnostics, sustainable agriculture, and transformative biotechnology. By sharing expertise across disciplines—from molecular biology, artificial intelligence, materials science, clinical medicine, and beyond—new innovations that will overcome both current and future challenges will be discovered.
Tomas Urbaitis, PhD, is senior scientist and Karolina Makovskytė is head of business development at Caszyme.
