Abstract The in utero electroporation (IUE) platform provides a rapid and flexible system to generate genetically and regionally diverse murine brain tumor models. Prior IUE brain tumor models have utilized CRISPR-Cas9 plasmids to target tumor suppressor genes, such as Trp53, to promote tumor formation and growth. Plasmid based CRISPR-Cas9 methods are highly useful, but cloning individual sgRNAs and validating their efficacy to induce gene disruption limits the flexibility of the interchangeable IUE system. Moreover, for applications examining non-tumor suppressor genes in IUE brain tumor models using CRISPR-Cas9, a high insertion-deletion (INDEL) efficiency would be needed to ensure tumor homogeneity for data interpretation. To improve the versatility and ease of using CRISPR-Cas9 in the IUE system, the utilization of mRNA based Cas9 and sgRNAs was optimized. Commercially available, “off the shelf,” Cas9 mRNA and synthetic sgRNA reagents generate high INDEL rates in a reproducible manner. Using multi-sgRNAs as a pool together to delete small fragments of a gene target is efficacious and has been shown to work more powerfully than single sgRNAs. Combining this technique with plasmid DNAs that promote tumorigenesis, the system can target genes to study their function in the context of tumor development and pathogenesis. This can include examining how the manipulation of specific tumor cell genes can alter interactions with the tumor microenvironment and immune response. Overall, this method can be added to the IUE toolbox, providing a more streamlined, reliable, and efficient way to target genes of interest in normal neurodevelopment and brain tumorigenesis.
Reel et al. (Fri,) studied this question.