Figure 5.

In utero delivery of Cas9 mRNA/gRNA with ADP-LNPs results in widespread editing of multiple neural cell populations. (A) Schematic diagram describing the experimental protocol used to determine the cell types edited by ADP-LNPs with Cas9 mRNA and gRNA. 96 h after in utero delivery, cell sorting of td-Tomato-positive cells was performed and the edited cells were analyzed via single-cell sequencing. (B) Flow cytometry analysis of the entire fetal brain at 96 h after in utero ICV injection. (C) Quantification analysis of the efficiency of in utero gene editing. At the Cas9/gRNA ratio of 1:3, 8.56 ± 2.23% of total brain cells were edited by a single injection (data are represented as mean ± SEM, n = 3). (D) Representative brain section images display the distribution of td-Tomato expression, particularly within the SVZ (b) and GE (c). (d) H&E staining of an adjacent coronal section showing the overall brain structure for comparison. (E) Detailed immunohistochemistry in the SVZ and GE regions reveals that td-Tomato expression colocalizes with the NSPC markers Sox2, Nestin, and Ki67. ADP-LNPs were able to edit NSPCs with Cas9 mRNA and gRNA. (F) The edited cells were sorted and the cell types edited were identified via single-cell sequencing. t-SNE analysis of single-cell sequencing data illustrates the diverse neural cell populations edited by ADP-LNPs containing Cas9 mRNA and gRNA. (G) Pie chart summarizing the proportions of different cell types expressing td-Tomato, indicating a wide range of edited cells, with notable fractions of neuron-related cells like interneurons, neuroblasts, and glioblasts.