Fig 1: Illustration of hybrid BV vector (Bac-LECW) carrying the CRISPRa system for neurotrophic factor gene activation. Bac-LECW harbored dCas9 (with 5' and 3' NLS sequences) fused with VP64 activation domain (dCas9-VP64) and the MPH (MCP-p65-HSF1) activation effector. dCas9-VP64 and MPH were linked with P2A sequence and co-expressed under the control of rat EF-1a promoter. Bac-LECW also harbored 9 sgRNA expression cassettes each consisting of the hU6 promoter, sgRNA 2.0 backbone with two MCP binding motifs and the spacer sequences targeting different sequences on the neurotrophic factor genes (BDNF: -75, -138 and -208; GDNF: -113, -225 and -466; NGF: -155, -186 and -691). The entire cassettes were flanked by two loxP sites. Co-transduction of ASC with Bac-Cre and Bac-LECW would lead to prolonged expression of dCas9-VP64 and 9 different sgRNA, which coordinate to target different positions of BDNF, GDNF and NGF genes and recruit the MPH effector to activate gene expression.
Fig 2: Confirmation of prolonged gene activation in ASC transduced by the hybrid BV and preparation of BV-transduced ASC sheets. (A-C) Expression profile of BDNF (A), GDNF (B) and NGF (C). (D) Fabrication and transduction of rat ASC sheets. The data represent means±SD of 3 independent culture experiments.
Supplier Page from RayBiotech for Rat beta-NGF ELISA