In the human gut, trillions of bacteria and their viral predators—phages—are locked in an ancient arms race. Bacteria deploy CRISPR-Cas immune systems to snip phage DNA, while phages counter with anti-CRISPR (Acr) proteins that block this defense. Despite the gut's status as a hotspot for microbial interactions, the diversity of Acrs encoded by gut phages is largely unexplored.

A study published in Cell Host & Microbe on March 18 and led by Prof. MA Yingfei from the Shenzhen Institute of Advanced Technology of the Chinese Academy of Sciences uncovered a vast repertoire of Acrs from human gut phages. It identified 651 potential Acrs targeting type II CRISPR systems, which dominate the gut bacterial landscape, and discovered a novel family of structurally convergent, dual-function Acrs named GutAcr_aca.

Researchers analyzed nearly 300,000 gut bacterial genomes and 33,000 phage genomes. They found that type II CRISPR systems account for 47% of all CRISPR systems in the gut, far outnumbering other types.

Focusing on six major Cas9 orthologs (SpyCas9, SaCas9, St1Cas9, St3Cas9, NmCas9, FnCas9), researchers identified ~4,500 Acr candidates through a guilt-by-association strategy, which were then cloned into a high-throughput functional screening library. After multiple rounds of CRISPR selection pressure, 651 Acrs were enriched, and 36 were confirmed to strongly inhibit CRISPR activity in plaque assays.

Moreover, researchers found that 213 of these Acrs, named GutAcr_aca, exhibited striking structural similarity despite minimal sequence homology. Most of them contained a helix-turn-helix motif, suggesting a dual role in transcriptional regulation.

Functional assays revealed that GutAcr_aca proteins could both autorepress their own transcription and block Cas9 through distinct mechanisms: GutAcr_aca-747 bound the PAM-interacting domain of SpyCas9, preventing DNA target recognition; GutAcr_aca-4292 and -2778 bound the sgRNA, disrupting Cas9-sgRNA complex formation. Notably, a 28-amino-acid peptide derived from GutAcr_aca-747 retained full inhibitory activity, making it the smallest known CRISPR inhibitor to date.

These GutAcr_aca proteins are widely distributed. They have been found in 26% of gut microbial species, and are especially enriched in temperate phages (82.6%), indicating their crucial role in phage lysogeny and gut ecosystem dynamics.

This work unveils the human gut virome as a rich source of Acr proteins, providing both ecological insights and biotechnological tools. The compact size and diverse mechanisms of the Acrs offer new opportunities for precision genome editing, microbiome engineering, and phage therapy.