Journal: Cell Reports Methods

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

doi: 10.1016/j.crmeth.2025.101299

Figure Lengend Snippet: Multiplex orthogonal gene regulation and programmable editing by OREC with therapeutic application in osteoblasts (A) Experimental illustration of orthogonal activation and repression of ASCL1 and RAB7A gene expression in HEK293T cells using distinct chemical and light inductions. (B) RT-qPCR time course analysis demonstrating orthogonal and reversible control of ASCL1 activation and RAB7A repression in response to alternating Dox and light stimuli. Data represent mean ± SEM ( n = 3). vs. RAB7A baseline (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001); vs. RAB7A 36 h ( $ p < 0.05, $$$ p < 0.001); vs. ASCL1 baseline ( && p < 0.01, &&&& p < 0.0001); vs. ASCL1 48 h ( ## p < 0.01, #### p < 0.0001). (C) Schematic diagram illustrating crRNA guide-length-dependent transcriptional repression and HDR-based gene editing using OREC c system. (D) Transcriptional repression of RAB7A and FZD1 genes following 72 h doxycycline treatment using 15 bp crRNA guides. Data represent mean ± SEM ( n = 3) (∗∗∗∗ p < 0.0001). (E) Luciferase activity analysis following HDR-mediated luciferase knockin using 20 bp crRNA guides. Data represent mean ± SEM ( n = 3) (∗∗∗∗ p < 0.0001). (F) Flow cytometry analysis showing percentage of GFP-positive cells following HDR-mediated GFP knockin. Data represent mean ± SEM ( n = 3) (∗∗∗∗ p < 0.0001). (G) Schematic representation illustrating simultaneous orthogonal activation of Bmp2 and repression of Dkk1 expression in MC3T3-E1 cells. (H) RT-qPCR analysis demonstrating significantly enhanced Bmp2 activation and Dkk1 repression under simultaneous orthogonal induction compared with single-gene regulatory conditions. Data are presented as mean ± SEM ( n = 3). ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (I) Representative images of alizarin red S (ARS) and alkaline phosphatase (ALP) staining showing significantly enhanced osteogenic differentiation following orthogonal OREC induction compared to single-gene controls. Color code for experimental conditions: white: empty vector control; gray: OREC o construct only; crosshatched: OREC c construct only; red: OREC o + Light (optogenetic activation); green: OREC c + Dox (chemogenetic activation) (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (J) RT-qPCR analysis demonstrating synergistic upregulation of osteogenic marker genes ( Alp , Bglap , and Sp7 ) upon simultaneous Bmp2 activation and Dkk1 repression. Data represent mean ± SEM ( n = 3) (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (K) The CCK-8 assay quantifies viable cells via dehydrogenase-mediated formazan formation measured at 450 nm, showing that OREC treatments do not affect cell viability in MC3T3-E1 cells. Data shown as mean ± SEM ( n = 3). See also and .

Article Snippet: Recombinant BMP2 protein , MedChemExpress , Cat# HY-P7006.

Techniques: Multiplex Assay, Activation Assay, Gene Expression, Quantitative RT-PCR, Control, Luciferase, Activity Assay, Knock-In, Flow Cytometry, Expressing, Staining, Plasmid Preparation, Construct, Marker, CCK-8 Assay

Journal: Cell Reports Methods

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

doi: 10.1016/j.crmeth.2025.101299

Figure Lengend Snippet: AAV-delivered OREC system design and in vivo orthogonal gene regulation in fracture healing (A) OREC o system comprises two vectors encoding dAsCpf1-Activer (upper) and TetR-CIBN-P2A-CRY2-VP16 3 with Bmp2 -targeting crRNA (lower). (B) OREC c system consists of vectors encoding dLbCpf1-Repr (upper) and another vector expressing rtTA-Advanced with Dkk1 -targeting crRNA (lower). See also . All constructs designed within AAV packaging constraints. (C) Experimental setup showing AAV intratibial injection procedure with custom LED patch device and power supply system for localized light delivery. (D) Experimental timeline: AAV intratibial injection 14 days before fracture (day 14), tibial fracture induction (day 0), and blue light treatment (470 nm, twice daily for 30 min) beginning one day post-fracture (day 1) through endpoint (day 42). (E and F) AAV2/9 vector constructs for bioluminescence analysis of OREC activity in vivo . (G) Experimental timeline showing AAV intratibial injection (day 0) followed by 14-day treatment with light stimulation (470 nm, 30 min twice daily) or oral doxycycline (30 mg/kg daily) prior to bioluminescence imaging. (H) Representative bioluminescence images demonstrating inducible luciferase expression at tibial sites by OREC with no detectable signal in distant tissues. (I) RT-qPCR analyses of Bmp2 and Dkk1 expression levels in orthogonally treated fracture sites at days 7 and 21 post-fracture. Data represent mean ± SEM ( n = 6 mice/group) (∗∗ p < 0.01, ∗∗∗∗ p < 0.0001). (J) Analysis of Bmp2 and Dkk1 expression relative to Gapdh using the 2 −ΔCt method in fracture callus tissue 7 days after light or doxycycline induction in OREC-transduced mice. Data represent mean ± SEM ( n = 6 mice per group). (K and L) RT-qPCR analysis of Bmp2 (K) and Dkk1 (L) expression in fracture callus tissue from mice exposed to inducers without OREC constructs. Data represent mean ± SEM ( n = 3 mice per group). See also and .

Article Snippet: Recombinant BMP2 protein , MedChemExpress , Cat# HY-P7006.

Techniques: In Vivo, Plasmid Preparation, Expressing, Construct, Injection, Activity Assay, Imaging, Luciferase, Quantitative RT-PCR