DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism

Dongmei Tang; Tingting Jia; Yongbo Luo; Biqin Mou; Jie Cheng; Shiqian Qi; Shaohua Yao; Zhaoming Su; Yamei Yu; Qiang Chen

doi:10.1016/j.xinn.2023.100495

DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism

Innovation (Camb). 2023 Aug 9;4(5):100495. doi: 10.1016/j.xinn.2023.100495. eCollection 2023 Sep 11.

Authors

Dongmei Tang¹, Tingting Jia², Yongbo Luo², Biqin Mou³, Jie Cheng², Shiqian Qi¹, Shaohua Yao², Zhaoming Su², Yamei Yu², Qiang Chen²

Affiliations

¹ Department of Urology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
² Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
³ Precision Medicine Center, Precision Medicine Key Laboratory of Sichuan Province, West China Hospital, Sichuan University, Chengdu 610041, China.

Abstract

In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.