CRISPRa assay was conducted using the SAM system ( The sgRNA for hLMR1 was designed using the following sequence: forward, CACCGGACAGACAGGAGAGCAGACT; reverse, AAACAGTCTGCTCTCCTGTCTGTCC. Briefly, the template was ligated into the sgRNA (MS2) cloning backbone (Addgene, 61424) using Golden-Gate reaction (SAM) after Golden-Gate annealing. The expression cassettes of dCas9-VP64 (Addgene, 61422) and MS2-P65-HSF1 (Addgene, 61423) were subcloned into pAdv5 vector (Invitrogen) for virus packaging. SgRNA elements with the U6 promoter were amplified and subsequently cloned into pAd/PL adenovirus vector (Invitrogen) for virus packaging. Viruses were amplified, desalted, and titered as described above. Three adenoviruses (1:1:1) were delivered into humanized mice intravenously at a total of 5 × 108 PFU/mouse. After 7 days, tissue samples were harvested for further analysis after mice were under food withdrawal for around 5 hours (from ~9:00 am to 2:00 pm).

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