Ancestral sequence reconstruction has led to the discovery and synthesis of ReChb, a revolutionary variant of CRISPR–Cas12a with remarkable versatility. ReChb’s ability to target any nucleic acid with minimal constraints positions it as a transformative tool for genome editing and genetic diagnostics.
CRISPR–Cas systems act as antiviral defense mechanisms in bacteria, where a Cas-family nuclease cuts specific sequences in viral genetic material. Engineered CRISPR–Cas systems have become widely used for gene editing and molecular diagnostics. However, natural nucleases, primarily sourced from bacteria have inherent molecular characteristics and limitations that prevent their full potential as therapeutic tools. For instance, Cas nucleases require a guide RNA to locate their target sequences but also depend on recognizing a protospacer-adjacent motif (PAM), a specific sequence adjacent to their targets. This dependency complicates the use of these enzymes for any desired therapeutic purpose.
To overcome these limitations, researchers worldwide have undertaken intensive efforts to develop CRISPR–Cas variants with broader and more adaptable applications.
The researchers hypothesized that reviving an ancient version of CRISPR–Cas12a one that had not yet evolved, could reveal fewer limitations in recognizing target sequences and substrates. This idea led to the development of ReChb (resurrected Cas from Hydrobacteria).
After reconstructing ReChb in the lab, the team conducted experiments without imposed restrictions using various PAM sequences, substrates, and guide RNAs. The results demonstrating that ReChb is highly versatile and operates with minimal constraints. This extensive targeting capability makes ReChb a versatile nuclease with numerous applications in biotechnology.
In conclusion, ReChb can be described as a versatile and all-purpose nuclease capable of targeting any nucleic acid. It is versatile because it exhibits both target-specific and non-specific collateral cutting activity. It is all-purpose because it functions with both single-stranded and double-stranded DNA and RNA. Furthermore, it can accommodate a wide range of guide RNAs from various species and displays significant adaptability in recognizing PAM sequences. With these characteristics, ReChb has the potential for applications in both gene editing and molecular diagnostics. Additionally, like other Cas nucleases, the size of
ReChb remains a limiting factor for delivery.
These challenges are significant in the development of new synthetic RNA-guided nucleases for therapeutic applications. It may be valuable to explore smaller nucleases as potential alternatives to overcome delivery issues. This study may also pave the way for exploring the global evolution of other RNA-guided nucleases, including Cas nucleases, and others, as well as their molecular relationships and potential applications in biotechnology.
By Miriana Braghin, Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, University of Milan.