Advancing brain health research through male germline editing in marmosets

This collaborative research will combine skills to optimize methodology to facilitate germline transmission of edited ARX alleles and to generate genetic tools useful to all marmoset researchers. including germline transmitted germ cell and neuronal reporter transgenes. and neuronal Cre drivers via the following three specific aims: 
 
 Neurological disorders have seen a dismal rate of new therapies despite promising findings in rodents. arguably due to problems translating these findings to the clinic. The human cortex features larger superficial layers. and more numerous and elaborate inhibitory interneurons than in rodents. The marmoset six-layered cortex mimics this anatomy. offering an ideal model for studies of neurodevelopmental and cognitive processes. However to fully exploit this model. there is a clear need for further development of genetic tools. Building on our long-standing interest in mutations in the Aristaless-related homeobox (ARX) gene. which can result in a structurally normal brain with epilepsy and intellectual disabilities. plus our extensive experience with primate germ and pluripotent cells. we propose to use CRISPR to create germline transmissible marmoset models of human ARX mutations. Transgenic marmosets have been generated by lentiviral or CRISPR/Cas9 methods via embryo injection. but this method is animal and surgery intensive. inefficient. expensive. subject to random chimerism. and validation of transgenesis cannot be performed until later in pregnancy or postnatally. We propose a novel approach using CRISPR to edit the genome of marmoset pluripotent stem cells (cjPSCs). that we can then differentiate into male germ cells that can be grafted or transplanted into testes where they can seed spermatogenesis in vivo to generate transgenic sperm and offspring. Our combined laboratories have extensive experience with nonhuman primate (NHP) PSCs and germ cells. the use of CRISPR to introduce transgenes into safe harbor loci in NHP genomes. and the production of functional. donor-derived sperm following germ cell transplantation into NHP testes. 
 
 Aim 1: Use CRISPR editing to generate mutant ARX alleles and reporter transgenes in cjPSCs. We will generate two types of ARX mutations: 1) a deletion predicted to cause lissencephaly and lamination defects and 2) a poly-alanine (pAla) expansion associated with interneuron defects. epilepsy and intellectual disability. Additionally. to enhance the utility of the male germline-mediated approach to produce transgenic marmoset offspring for neurodevelopmental research. we will use CRISPR to generate cjPSC lines carrying reporter transgenes expressed from neuronal- or germ cell-specific promoters. along with neuronal Cre drivers to provide new tools for conditional genetic modifications in marmosets. The use of both fluorescent and MRI reporter systems will maximize the potential for non-invasive imaging of transgenic cells. 
 
 Aim 2: Optimize derivation and transplantation of male cjPSC-sourced germ cells into recipient testes. 
 Leveraging our close proximity to one of the world???s largest breeding colonies of marmosets at the Southwest National Primate Research Center. we will optimize protocols for deriving male germ cells from CRISPR-edited cjPSCs that can be used to produce transgenic marmoset sperm in vivo. We will determine which of four alternate pathways will yield optimal donor-derived spermatogenesis from in vitro-differentiated germ cells. including germ cell transplantation into newborn or adult germline-depleted testes. homotopic or orthotopic reconstituted testis grafting. or xenografting into mice. The goal will be to validate the method that most efficiently facilitates production of otherwise normal transgenic marmoset offspring from CRISPR-edited sperm. Normalcy of gene expression and epigenetic programming in cjPSC-derived male germ cells (as compared to corresponding. wild-type endogenous male germ cell types) will be assessed by histology. immunohistochemistry. and single-cell (sc) or bulk profiling of gene expression (scRNA-seq). chromatin accessibility (scATAC-seq). DNA methylation (whole-genome bisulfite sequencing) and histone modifications (ChIP-seq).
 
 Aim 3: Assess the impact of ARX mutations on marmoset cortical neuron development and migration.
 CRISPR-edited cjPSCs will be used to assess the impact of ARX deletion or pAla-expansion on cortical neuron development and interneuron migration in cortical and subpallial organoids. cjPSC-derived marmoset neurons will be assessed by immunohistochemistry and scRNA-seq to facilitate comparisons to corresponding endogenous neurons from fetal and postnatal marmoset brains to characterize the proportions of different cortical cell types. interneuron migration in the context of primate neuroanatomy. and normality of neuronal subtype-specific gene expression and epigenetic programming. 
 
 Taken together. the proposed research is designed to develop a novel. reliable an