Molecular Mechanisms Behind Type V CRISPR Identified

Type V CRISPR-Cas, also known as Cas12, has become one of the most effective tools for genome editing in basic research, medicine, and agriculture, but how it emerged has been unclear. Now, a team led by Professor Gao Caixia from the Institute of Genetics and Developmental Biology at the Chinese Academy of Sciences has revealed a fundamental molecular innovation underlying the origin of Type V CRISPR-Cas immune systems.

Published in Cell, their study reveals that the functional splitting of transposon-derived RNAs marked a pivotal step in the development of Type V CRISPR-Cas immunity. While earlier investigations recognized TnpB nucleases encoded by IS200/605 transposons as ancestral proteins for the Type V Cas12 effectors, the mechanisms linking transposon activity to CRISPR immune function were previously ambiguous. 

To elucidate the origins of Type V systems, the research group adopted a mining strategy focused on catalytic motifs, structural domains, and sequence similarity shared between TnpB nucleases and Cas12 effectors. Expansive searches of prokaryotic genomes and metagenomic repositories uncovered 146 distinct TnpB-like CRISPR-associated proteins. Subsequent phylogenetic analyses, AlphaFold structural predictions, and comparisons of functional elements led to the identification of six intermediate clades, termed TranCs, which are sister groups to particular TnpB lineages. Clades 3, 11, 12, 13, and 14 trace their origins to IS605 transposons, while clade 8 (Cas12n) derives from IS607, representing essential evolutionary intermediates between TnpB and Cas12.

Functional assays disclosed that TranCs possess a unique dual-guide RNA mechanism. Five TranC systems utilize their own CRISPR RNAs (tracrRNA-crRNA hybrids) for DNA targeting, while maintaining the ancestral ability to employ transposon-derived reRNAs (also referred to as ωRNAs) for directing DNA cleavage. This dual-guidance trait marks TranCs as functional intermediates in the transition from TnpB nucleases to Cas12-based CRISPR immunity.

Cryo-EM analysis demonstrated a notable similarity between the LaTranC-sgRNA-DNA complex and the ISDra2 TnpB-reRNA-DNA complex—with the crucial difference being the RNA's division into tracrRNA and crRNA. Experimental RNA splitting of TnpB’s reRNA successfully transformed TnpB into a CRISPR-like nuclease capable of using CRISPR arrays as sources of guide RNAs.

The study highlights that innovation at the RNA level, not drastic changes in protein structure, drove the emergence of Type V CRISPR-Cas systems. Additionally, the identification of compact nucleases with adaptable guide RNAs provides principles for designing genome editing tools that are both smaller and simpler to control.