Using AI Tools to Uncover the Mystery of CRISPR-Cas13 System Evolution

The CRISPR-Cas system is an adaptive immune system of bacteria and archaea that can resist the invasion of viruses and mobile genetic elements. Although the DNA targeting mechanisms of CRISPR-Cas9 and CRISPR-Cas12 systems have been widely studied, the evolutionary origin of the RNA-targeted CRISPR-Cas13 system is still unclear.

In February 2025, Zhang Feng's team published a research paper entitled "Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins" in the top international academic journal Cell. The study revealed that the RNA-targeted CRISPR-Cas13 system may have originated from an ancient RNA toxin-antitoxin (TA) system, especially the AbiF system, through a hybrid structural and sequence evolution tracking method. This study not only provides new insights into the evolution of CRISPR systems, but also provides important clues for understanding the diversity of RNA-guided mechanisms in the immune system.

The CRISPR-Cas13 system contains two nuclease domains of the HEPN (higher eukaryotic and prokaryotic nucleotide binding) superfamily, which are widely present in the defense systems of prokaryotes.

In this latest study, the research team identified the ancestor of Cas13 through hybrid structure and sequence search, predicted protein structure through deep learning tools such as AlphaFold2, and combined with algorithms such as DALI for structural similarity comparison, and found that Cas13 may have originated from the AbiF system. AbiF (Abortive Infection Protein F) is a toxin-antitoxin system (TA) encoded by genes related to abortion infection. Its antitoxin is a non-coding RNA (ncRNA) that can inhibit the activity of RNA toxins.

The research team also discovered an intermediate system, Cas13e, which has the biochemical characteristics of both CRISPR and TA systems. Cas13e is an RNA-guided RNA targeting system, while AbiF is a TA system with RNA antitoxins. The mechanism of action of the AbiF system is different from that of the CRISPR system. The ncRNA antitoxin inhibits the activity of AbiF by blocking its nuclease active site. The evolutionary status of Cas13e is between AbiF and other known Cas13s.

The research team further verified the function of Cas13e and found that it can bind to crRNA and has RNA-guided RNA cleavage activity. Cas13e exhibits non-specific RNA cleavage activity after binding to crRNA, similar to the Cas13a/b/c/d system.

Through sequence and structure comparison, the research team reconstructed the evolutionary path from AbiF to Cas13. The AbiF gene first replicated, producing a dual HEPN nuclease with an additional domain insertion. The replicated protein gradually lost its interaction with the cis-encoded ncRNA antitoxin and instead interacted with CRISPR RNA (crRNA), eventually evolving into an RNA-guided CRISPR system.

That is, AbiF, as a toxin-antitoxin (TA) system protein associated with the abortive infection mechanism, forms a stable complex with non-coding RNA (ncRNA) as a primitive defense system to prevent the spread of infection (such as phage infection) by inducing host cell dormancy/death. The CRISPR-Cas13 system has evolved programmable RNA targeting capabilities on its basis. Based on these findings, the research team proposed an evolutionary model that reveals how non-RNA-guided toxin nucleases (AbiF systems) evolved into RNA-guided CRISPR systems (CRISPR-Cas13 systems).

Figure 1. the key structural changes enabled a non-guided TA system to evolve into an RNA-guided CRISPR system. (Zilberzwige-Tal S, et al., 2025)

These findings suggest that the evolutionary path of Cas13 appears to be a unique event, unlike the CRISPR-Cas9 and Cas12 systems. Although AbiF interacts with ncRNA, it does not use ncRNA guidance, so the transition from AbiF to RNA-guided CRISPR systems is more challenging. This study reveals how RNA-guided mechanisms evolved from non-RNA-guided toxin nucleases, broadening our understanding of the diversity of prokaryotic RNA immune systems.