Lizards are able to regrow amputated tails, but the lizard tail regenerative process fails to recapitulate the dorsoventral patterning achieved during embryonic tail development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. Embryonic NTs are, themselves, dorsoventrally patterned, with Pax7+ Shh- dorsal roof plate domains that restrict cartilage skeletal formation induced by Pax7- Shh+ floor domains to ventral tail regions. However, adult regenerated tail ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tube skeletons. Both NTs and ETs contain populations of neural stem cells (NSCs), but only embryonic NSC populations are able to differentiate into roof plate identities and neurons. Embryonic NSCs transplanted into regenerated tail ETs retain the capacity to form roof domains but are ultimately ventralized by the unchecked hedgehog signaling of regenerated lizard tail environments. We hypothesized that only the simultaneous repression of hedgehog signaling and enhancement of NCS roof plate differentiation capacity would induce patterning in lizard ETs and, hence, regenerated cartilage. This was tested through the use of a novel genetic engineering process in which NSCs are isolated from embryos of the parthenogenetic lizard Lepidodactylus lugubris, gene-edited in vivo, and implanted back into clonally-identical adults to regulate tail regeneration. Embryonic lizard NSC lines unresponsive to hedgehog stimulation were generated through the use of CRISPR/Cas9 technologies to knockout (KO) the signaling regulator smoothened (Smo). Exogenous Smo KO NSCs were injected into adult tail spinal cords, where they engrafted to endogenous ependymal cell populations and contributed to dorsal domains in regenerated tail ETs. Embryonic Smo KO NSCs maintained roof plate identities in vivo, and lizards treated with edited NSCs regrew tails that lacked cartilage in dorsal regions. These studies represent an important milestone in the creation of the first regenerated lizard tails with dorsoventrally patterned ETs and skeletal tissues.