Fig. 4: CRISPRa of maternally silenced X genes Sash3, Tlr7 and Cysltr1 improves cognition in old female mice.

a, CRISPRa⁺ lentiviral plasmids (dCas9 + sgRNA constructs), experimental model and timeline of in vitro validation of CRISPRa⁺ cells. Scr, scrambled sgRNA. b, Validation using primary neuronal cultures showed CRISPRa lentivirus mediates increased mRNA expression of Sash3 (*P = 0.0049, n = 6 wells per group), Tlr7 (*P = 0.0019, n = 6 wells per group) and Cysltr1 (*P = 0.0429, n = 7 wells control, n = 5 wells treatment). Unpaired two-tailed t-tests. c, CRISPRa lentivirus (dCas9 + sgRNAs) was stereotaxically injected into the hippocampus of female C57BL/6J mice (n = 14 mice per group, age: 18 months). Hippocampi were collected for immunohistochemistry (IHC) and RNA-seq. d, Representative dentate gyrus image following stereotaxic injection of dCas9 (red) and sgRNA (green) lentivirus. Scale bar, 50 µm. DAPI (blue), nuclei. White arrows indicate co-labelling of dCas9 and sgRNA. n = 3 mice imaged. e, Percentage of dentate gyrus cells expressing sgRNA (green), dCas9 (red) and yellow (co-localization of sgRNA and dCas9) (n = 3 mice). f, Transfected hippocampi were FACS-sorted to obtain GFP⁺ (sgRNA) neurons (NeuN⁺) in control and CRISPRa⁺ groups for RNA-seq. g, Heat map of top 100 DEGs following RNA-seq of control compared with CRISPRa⁺ hippocampi (age: 22 months; n = 4 samples per group). h, Gene ontology analysis of DEGs comparing control and CRISPRa⁺ hippocampal samples (Fisher’s exact test, PANTHER GO analysis). i, Experimental model and timeline of behavioural testing in old female mice. MWM, Morris water maze; NPR, novel place recognition. j, Spatial learning in the hidden trials, measured as the distance travelled to find the platform, shows that CRISPRa⁺ overexpression of Sash3, Tlr7 and Cysltr1 improved learning compared with controls (control mice: n = 14–15; CRISPRa⁺ mice: n = 13–14 mice; age: 20 months). Two-way mixed-model ANOVA: treatment, *P = 0.0154. k, Probe trials show that CRISPRa⁺ improved memory after hidden training (n = 14 mice per group, age: 20 months). Unpaired two-tailed t-test, *P = 0.0054. l, CRISPRa⁺ in mice improved spatial memory measured as the increased time spent with the object at the novel position, compared with control mice (control mice: n = 11; CRISPRa⁺ mice: n = 12). Unpaired two-tailed t-test, *P = 0.0233. Data represent mean ± s.e.m.

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