DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.

Effectiveness and safety of community-led assisted partner service among HIV-diagnosed men who have sex with men: a multicentre, randomized controlled trial in China.

Background: No randomized controlled trials have involved established HIV-diagnosed men who have sex with men (MSM) diagnosed for more than 6 months into the assisted partner service (aPS). We compared voluntary aPS involving community-based organizations (CBOs) and HIV self-testing (aPSST) with regular partner service (rPS) in HIV-diagnosed MSM irrespective of diagnosis time. Methods: In this unblinded, multicentre trial, we enrolled HIV-diagnosed MSM irrespective of diagnosis time in three cities in northern China. Index patients were randomly assigned to aPSST or rPS. Index patients in the aPSST group were additionally provided a comprehensive intervention package including HIV self-testing and CBO-based aPS compared with rPS group. The primary outcome was the number of index patients whose any sexual partner tested for HIV during the 6-month study. Completion of HIV testing was defined as sexual partners taking a clinic-based HIV test or HIV self-testing. Safety was assessed preliminary at the end of the 6-month follow-up. This study has been registered at chictr.org.cn (ChiCTR2000038784). Findings: From March to December 2021, 325 of HIV-diagnosed MSM were enrolled (90⋅2% were established HIV-diagnosed MSM) and randomly assigned to receive aPSST (n = 167) or rPS (n = 158). At 6 months, 110 (65⋅9%) index patients in the aPSST group had at least one sexual partner tested for HIV compared with 50 (31⋅6%) in the rPS group (hazard ratio 2⋅86; 95% confidence interval 2⋅03-4⋅03; p < 0⋅001). No significant difference was observed in effects of aPSST on HIV testing promotion between established and newly HIV-diagnosed MSM. Self-reported harms were infrequently observed in both groups (approximately 2⋅0%). Interpretation: Among HIV-diagnosed MSM regardless of diagnosis time, voluntary aPS involving CBOs and HIV self-testing was effective and safe for promoting partner HIV testing. Funding: This work was supported by the Mega-Projects of National Science Research, the National Natural Science Foundation of China and the Liaoning Revitalization Talents Program, China.