Virus and shRNA generation

AAV was produced by transfection of 293 cells with three plasmids: an AAV vector expressing target constructs (EmGFP, EmGFP-Lepr shRNA, DIO-EmGFP, DIO-EmGFP-Lepr shRNA, saCas9, Lepr sgRNA (a gift from D. Kong), DIO-GCaMP6f, DIO-eGFP, DIO-Synaptophysin-eGFP, DIO-eNpHR3.0-eYFP, DIO-eYFP, fDIO-mCherry, fDIO-hM3D-mCherry, fDIO-hM4D-mCherry, fDIO-eGFP, fDIO-Kir2.1-eGFP, DIO-mCherry, DIO-mCherry-hM3D, DIO-mCherry-hM4D and DIO-mRuby2-TVA-RVG), AAV helper plasmid (pHELPER; Agilent) and AAV rep-cap helper plasmid (pRC-DJ, a gift from M. Kay). At 72 h after transfection, the cells were collected and lysed. Viral particles were then purified by an iodixanol step-gradient ultracentrifugation method. The iodixanol was diluted and the AAV was concentrated using a 100-kDa-molecular-mass-cutoff ultrafiltration device. The genomic titer was determined by qPCR. The AAV vectors were diluted in PBS to a working concentration of approximately 10¹² viral particles per ml. To generate EnvA-pseudotyped glycoprotein (G)-deleted rabies virus expressing eGFP (RVΔG-eGFP), we followed a published protocol⁶⁸. Plasmids expressing the rabies viral components, B7GG, BHK-EnvA and HEK-TVA cells were provided courtesy of E. M. Callaway. HSV-DIO-Flp was purchased from the Gene Delivery Technology Core at the Massachusetts General Hospital.

To construct shRNA against Lepr ({"type":"entrez-nucleotide","attrs":{"text":"NM_146146.3","term_id":"1644264299","term_text":"NM_146146.3"}}NM_146146.3), oligonucleotides that contained 21-base-pair sense and antisense sequences (5′-AACTGATGAAGAGCAAGGGTT-3′) targeting Lepr were connected with a hairpin loop followed by a poly(T) termination signal. This shRNA oligonucleotide was ligated into BLOCK-iT POLII miR RNA-mediated interference expression vectors (Invitrogen) and then transferred to an AAV vector together with EmGFP. To test the efficacy of the shRNA, we stereotaxically injected AAVs expressing Lepr shRNA into the LH. Two weeks after injection, the virus-infected area labeled by EmGFP expression was dissected. Lepr mRNA levels were measured by qPCR and found to be reduced over 70% (Fig. ​(Fig.2d2d and Extended Data Fig. ​Fig.3l3l).