Size of nucleases determines their potential use
There are many CRISPR-Cas nucleases already identified, each with unique properties that make them suitable for various genome editing applications. Among these properties, the size of the nuclease plays a crucial role, particularly in the context of therapeutic applications. Cas nucleases range significantly in their size, from the compact Cas12f, consisting of approximately 400 amino acids, to the much larger nucleases such as LshCas9, which comprises of over 1500 amino acids. This size variation not only reflects their diverse functional capabilities but also directly influences their potential for practical use.
Large size of nucleases is one of the key limitations in the therapeutic context when it comes to delivery of gene editing tools to cells and tissues. One of the most commonly used delivery vector for therapeutic agents is Adeno-Associated Viruses (AAVs). AAVs are primarily chosen for delivery due to their low immunogenicity and broad tropism. However, AAVs have a limited packaging capacity, making it difficult to deliver nucleases exceeding 1100 amino acids. This constraint underscores the need for smaller nucleases, which can be efficiently packaged to AAVs, enabling more effective delivery to target sites. Moreover, recent research suggests that smaller nucleases may be less toxic and immunogenic compared to their larger counterparts, further highlighting their therapeutic potential.
The benefits of compact nucleases
At Caszyme we have extensive expertise working with small nucleases, including Cas12l and compact Cas9 variants, which we offer for licensing. Cas12l, with an effector size of approximately 850 a.a., is a small nuclease that stands out for its versatility. AspCas12l, in particular, works well across a broad temperature range, exhibits collateral ssDNA and ssRNA activity and functions effectively in a heterologous cellular environment.
Within Cas9 orthologs, proteins such as Cpe (1065 a.a.) and Msc (1044 a.a.) also qualify as small nucleases. These Cas9 variants are not only compact but also exhibit more specific PAM requirements compared to the widely used SpyCas9, which allows to expand the targeting range of CRISPR gene editing. Due to the compact size they are ideal candidates for AAVs packaging, making them more attractive for therapeutic applications in mammalian cells.
Taken together, small nucleases highlighted above are not only compact enough to be delivered via AAVs but have also demonstrated functionality in mammalian cells, showcasing their strong potential for clinical applications. As the demand for efficient and safe gene-editing tools in therapeutic contexts continues to grow, these small nucleases represent a promising solution, combining efficacy with the practical advantages of reduced size.