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. 2019 Aug 14;9(21):6099–6111. doi: 10.7150/thno.36790

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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-1α 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.