CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration

Abstract

Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.

Fluorescent PSC-Derived Cardiomyocyte Reporter Lines: Generation Approaches and Their Applications in Cardiovascular Medicine

“…Unlike nodal and ventricular types, the atrial PSC-CM markers are not fully characterised. Further insights from novel fluorescent atrial PSC-CM reporter lines, such as PITX2 and KCNA5 , in combination with atrial-promoting differentiation approaches including retinoic acid treatment and CRISPRa/CRISPRi-mediated transcriptional activation/inhibition of atrial-regulating pathways [ 136 , 137 ], could assist the development of atrial cell isolation protocols.…”

Genetically Engineered-MSC Therapies for Non-unions, Delayed Unions and Critical-size Bone Defects

“…Additionally, genome editing techniques like CRISPR/Cas9 hold great potential in the production of genetically modified MSCs. Recently, by repurposing the technique for CRISPR interference (CRISPRi), by using a catalytically inactive Cas9 (dCas9) which orchestrates with sgRNA to sterically block the transcription of target genes, Truong et al were able to simultaneously activate Sox9 and repress PPAR-γ in rat BM-MSCs [59]. Importantly, implantation of these CRISPRai-engineered BM-MSCs seeded into gelatin scaffolds improved the healing of critical-size calvarial bone defects in rats [59].…”

CRISPR Interference–Potential Application in Retinal Disease

“…The cells were then cultured into engineered cartilage and implanted into calvarial bone defects in rats. The CRISPR-untreated cartilage had negligible bone growth, while the CRISPR-treated cartilage saw significant bone repair, filling 28.6% and 23.3% of the original defect area [58].…”