An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair

Jie Zhao¹, Zengliang Wang², Lina Lu³, Guoyun Bu², Zukang Miao⁴, Yang Zhang⁵, Yue Guo⁵, Zhao Yang², Jianxiong Ma⁵, Jun Jiao⁶, Xinlong Ma⁷

Affiliations

¹ Department of Orthopedic, Tianjin Hospital, Tianjin 300211, China; Tianjin Hospital, Tianjin University, Tianjin 300211, China. Electronic address: zhaojie@tmu.edu.cn.
² Department of Orthopedic, Tianjin Hospital, Tianjin 300211, China.
³ Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, School of Medicine, Nankai University, Tianjin 300071, China.
⁴ Department of Orthopedic, Tianjin Hospital, Tianjin 300211, China; Tianjin Hospital, Tianjin University, Tianjin 300211, China.
⁵ Department of Orthopedic, Tianjin Hospital, Tianjin 300211, China; Tianjin Key Laboratory of Orthopedic Biomechanics and Medical Engineering, Tianjin Hospital, Tianjin 300050, China.
⁶ Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, School of Medicine, Nankai University, Tianjin 300071, China. Electronic address: junjiao@nankai.edu.cn.
⁷ Department of Orthopedic, Tianjin Hospital, Tianjin 300211, China; Tianjin Hospital, Tianjin University, Tianjin 300211, China; Tianjin Key Laboratory of Orthopedic Biomechanics and Medical Engineering, Tianjin Hospital, Tianjin 300050, China. Electronic address: maxinlong8686@yeah.net.

PMID: 41679307
DOI: 10.1016/j.crmeth.2025.101299

Abstract

CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: OREC^C regulated by doxycycline (Dox) and OREC^o controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC’s therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.

Keywords: CP: genetics; CP: molecular biology; CRISPR/Cpf1; chemogenetic; fracture healing; optogenetic; orthogonal.

Cell Rep Methods. 2026 Feb 23;6(2):101299.doi: 10.1016/j.crmeth.2025.101299. Epub 2026 Feb 11. (IF:4.500).

An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair

Abstract

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