RESUMEN
Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells.
Asunto(s)
Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Péptidos de Penetración Celular , Edición Génica , Edición Génica/métodos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/aislamiento & purificación , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/aislamiento & purificación , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/aislamiento & purificaciónRESUMEN
Color polymorphic species provide an excellent opportunity to investigate the ecology and evolution of intraspecific niche differences. The red-backed salamander, Plethodon cinereus, is a fully terrestrial lungless salamander with two common color forms, striped and unstriped. Previous research suggests the morphs may be differentially adapted to surface and subsurface microhabitats, with the unstriped morph being more fossorial. This hypothesis predicts that the unstriped morph should be more sensitive to the risks of surface activity (e.g., thermal stress, dehydration, predation), and therefore be more selective than striped morphs when choosing soil surface microhabitats. To test this hypothesis, we experimentally manipulated leaf litter mass in small forest patches (~0.45 m2). Leaf litter addition reduced soil temperatures, buffered against changes in air temperature, and likely provided physical protection from predators. Over 3 years, we found that unstriped adults responded positively to leaf litter addition, but striped adults did not. In addition, unstriped morphs spent significantly more time in protective refuges (opaque, moistened tubes) than striped morphs in laboratory assays. Taken together, the field and laboratory results support the hypothesis that the unstriped morph is more sensitive to the risks of surface activity, and therefore is more likely to be fossorial. This difference in microhabitat use, combined with spatiotemporal variation in leaf litter accumulation on the forest floor, may play an important role in the maintenance of the polymorphism.