Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.

CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.

Locked plating of distal femur fractures leads to inconsistent and asymmetric callus formation.

OBJECTIVES: Locked plating constructs may be too stiff to reliably promote secondary bone healing. This study used a novel imaging technique to quantify periosteal callus formation of distal femur fractures stabilized with locking plates. It investigated the effects of cortex-to-plate distance, bridging span, and implant material on periosteal callus formation. DESIGN: Retrospective cohort study. SETTING: One Level I and one Level II trauma center. PATIENTS: Sixty-four consecutive patients with distal femur fractures (AO types 32A, 33A-C) stabilized with periarticular locking plates. INTERVENTION: Osteosynthesis using indirect reduction and bridge plating with periarticular locking plates. MAIN OUTCOME MEASUREMENT: Periosteal callus size on lateral and anteroposterior radiographs. RESULTS: Callus size varied from 0 to 650 mm2. Deficient callus (20 mm2 or less) formed in 52%, 47%, and 37% of fractures at 6, 12, and 24 weeks postsurgery, respectively. Callus formation was asymmetric, whereby the medial cortex had on average 64% more callus (P=0.001) than the anterior or posterior cortices. A longer bridge span correlated minimally with an increased callus size at Week 6 (P=0.02), but no correlation was found at Weeks 12 and 24 postsurgery. Compared with stainless steel plates, titanium plates had 76%, 71%, and 56% more callus at Week 6 (P=0.04), Week 12 (P=0.03), and Week 24 (P=0.09), respectively. CONCLUSIONS: Stabilization of distal femur fractures with periarticular locking plates can cause inconsistent and asymmetric formation of periosteal callus. A larger bridge span only minimally improves callus formation. The more flexible titanium plates enhanced callus formation compared with stainless steel plates.