A phage DUF669 protein mediates post-cleavage DNA repair against host CRISPR immunity

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. Using affinity purification-mass spectrometry, these authors identified the Healer system, a phage two-protein effector that repairs CRISPR-mediated DNA breaks via homologous recombination.