Researchers have unveiled a critical discovery regarding Type V CRISPR-Cas systems, a powerful tool in genome editing that has significant implications for basic research, medicine, and agriculture. A groundbreaking study spearheaded by Professor Gao Caixia from the Institute of Genetics and Developmental Biology (IGDB) at the Chinese Academy of Sciences (CAS) uncovers a stunning truth—functional splitting of transposon-derived RNAs is perhaps the most important functional innovation that was critical in the evolution of Type V CRISPR-Cas immunity. Those shocking results were published Sept. 29 to the journal Cell.
Type V CRISPR-Cas systems, more often known as Cas12 systems, evolved from transposons. These transposons are DNA segments that can copy themselves into new locations within a genome. This unique evolutionary history is key to understanding how and why these systems operate as they do, and more importantly—to whom and what they are best applied. Based on phylogenetic analysis, TnpB nucleases, which are encoded by IS200/605 transposons, are the ancestral proteins. From these proteins, type V Cas12 effectors emerged.
Understanding Type V CRISPR-Cas Systems
Type V CRISPR-Cas systems are becoming more popular in the field, thanks to their versatility and efficiency in genome editing. For these reasons, these systems are of considerable value for basic genetic research as well as therapeutic development. By cutting out and replacing exact strands of DNA, they’re opening the door for revolutionary new treatment possibilities. The remarkable ability to edit genes has opened new avenues for research, furthering our understanding of genetic diseases and possible cures.
This work showcases that five TranC systems have used their natural intrinsic CRISPR RNAs, namely tracrRNA-crRNA hybrids for DNA targeting. This mechanistic insight is central to nearly all of these systems’ abilities to identify and cut targeted DNA sequences. By keeping an ancestral ability to use transposon-derived reRNAs, or ωRNAs, to help target DNA cleavage, these systems have evolved to very efficiently guide DNA cutting.
The research team identified 146 TnpB-like CRISPR-associated proteins by exploring prokaryotic genomes and metagenomic databases, shedding light on the evolutionary trajectories that shaped these systems. In fact, IS605 is the progenitor for clades 3, 11, 12, 13, and 14. Clade 8, or Cas12n, springs from IS607. This new classification forms a key evolutionary bridge between TnpB and Cas12.
“Discovery of TranC systems reveals the molecular mechanism of CRISPR origin.” – IGDB
The Evolutionary Pathway of CRISPR Systems
One unexpected discovery unearthed by this study was the conservation of RNA cleavage as a primary characteristic of Cas12 across three widely diverse clades. These clades are all descended from IS605 and IS607. This reaction cleaves often rather long RNAs into two resulting separate parts, tracrRNA and crRNA. These components are critical for safely and precisely targeting and slicing DNA strands.
A major component of this study focused on cryo-electron microscopy (cryo-EM) characterization of the LaTranC-sgRNA-DNA complex. When compared to the ISDra2 TnpB-reRNA-DNA complex this outcome was particularly surprising. This discovery only adds to evidence suggesting that RNA cutting is a central feature of the function of these CRISPR systems.
Insights from Structural Analysis
The only multi-functional reRNA observed, separated functionally. This process deeply enriches the hypothesis that functional splitting is key to the operation of Type V CRISPR-Cas systems. Such insights contribute to a deeper understanding of how these innovative tools have evolved over time and their potential for future applications.
The single reRNA observed underwent functional splitting, reinforcing the concept that this mechanism is foundational to the operation of Type V CRISPR-Cas systems. Such insights contribute to a deeper understanding of how these innovative tools have evolved over time and their potential for future applications.