Nature communicationsOct 22, 2026

A phage DUF669 protein helps fix DNA after cutting to resist host CRISPR defense

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Oct 23, 2026

Abstract

A phage-encoded system, termed Healer, neutralizes CRISPR immunity via a post-cleavage DNA repair mechanism.

Healer consists of two proteins: Gp63 and Gp64.
Gp63 binds to single-stranded DNA and facilitates the repair of CRISPR-induced DNA breaks.
Gp64 is involved in promoting homologous recombination for repairing phage DNA.
The Healer system demonstrated high phage genome-editing efficiency when co-expressed with Cas9/Cas12 in multiple bacterial species.
This finding suggests the existence of alternative anti-CRISPR mechanisms beyond previously known strategies.

Simplified

Phages have evolved various anti-CRISPR (Acr) proteins or RNA-based anti-CRISPR (Racr) to evade host CRISPR immunity by direct interference. However, whether other counter-CRISPR mechanisms exist remains unexplored. Here, we report a phage-encoded two-protein system, termed Healer, which neutralizes CRISPR immunity via a post-cleavage DNA repair mechanism. This phage replication-associated system comprises two proteins: Gp63 (a DUF669 domain-containing protein), and Gp64 (an AAA domain-containing protein). Mechanistic investigations elucidate that Gp63 acts as a rapid-response effector of CRISPR-induced DNA breaks. After Gp63 binds ssDNA, it promotes Gp64-mediated homologous recombination, which repairs CRISPR-generated phage DNA breaks and supports phage viability. Notably, co-expression of the Healer system with Cas9/Cas12 in E. coli, P. aeruginosa, and A. baumannii demonstrated high phage genome-editing efficiency. Collectively, our findings reveal an important anti-CRISPR complementary strategy, providing a promising tool for genome engineering.

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A phage DUF669 protein helps fix DNA after cutting to resist host CRISPR defense

Abstract

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