A Novel Method for In Vivo Gene Editing in the Brain of Guppies Using Unique Nanoparticles as Delivery Vehicles

Background Uncovering the genomic basis of traits has advanced rapidly in evolutionary biology and neuroscience, largely through phenotypic traits with adaptive value has been advancing rapidly in evolutionary biology, neuroscience, and behavior, largely due to research using non-traditional model systems. Direct gene editing in the adult brain represents a crucial next step in linking genotype to phenotype, avoiding the confounding effects that arise from modifications during development. However, implementing these technologies beyond traditional laboratory models remains challenging due to delivery limitations. Methods We developed an intracranial microinjection protocol for adult guppies (Poecilia reticulata) to deliver gene-editing elements to brain cells. We designed magnetic nanoparticles functionalized with a novel translocating agent, a non-viral carrier capable of transporting linearized nucleic acids across cellular and nuclear membranes. We comprehensively assessed nanoparticle uptake, nuclear colocalization, and potential health impacts using histological analysis, liver enzyme activity assays, and behavioral assessments. Results Our functionalized nanoparticles successfully entered brain cells and colocalized with nuclei at rates exceeding 50% after two weeks, demonstrating their potential for efficient in vivo gene editing. Health assessments showed no significant brain cell death (> 80% viability), no liver toxicity (normal ALT, AST, and ALP enzyme levels), and no alterations in individual and social behaviors, confirming the nanoparticles’ biocompatibility and systemic safety. Conclusions Our results, combined with previous in vitro work demonstrating our functionalized magnetic nanoparticles are an effective delivery system for gene editing, show they can be used for safe in vivo interventions in the adult brain of P. reticulata. This protocol overcomes a major technical barrier in evolutionary biology and neuroscience, with a novel nucleic acid-carrying vehicle that can be used in vivo in the adult brain. This approach provides a versatile platform for studying the genetic mechanisms underlying behavior in small freshwater fish while helping overcome the major limitations of conducting functional studies on non-model organisms.