CRISPR in butterflies: An undergraduate lab experience to inactivate wing patterning genes during development.

CRISPR is a technique increasingly used in the laboratory for both fundamental and applied research. We designed and implemented a lab experience for undergraduates to carry out CRISPR technology in the lab, and knockout the wing patterning genes optix and WntA in Vanessa cardui butterflies. Students obtained spectacular phenotypic mutants of butterfly wings color and patterns, awakening curiosity about how genomes encode morphology. In addition, students successfully used molecular techniques to genotype and screen wild-type caterpillar larvae and butterflies for CRISPR edits in genes. Student feedback suggests that they experienced a meaningful process of scientific inquiry by carrying out the whole CRISPR workflow process, from the design and delivery of CRISPR components through microinjection of butterfly eggs, the rearing of live animals through their complete life cycle, and molecular and phenotypic analyses of the resulting mutants. We discuss our experience using CRISP genome editing experiments in butterflies to expose students to hands-on research experiences probing gene-to-phenotype relationships in a charismatic and live organism.

"Designer babies?!" A CRISPR-based learning module for undergraduates built around the CCR5 gene.

CRISPR-cas technology is being incorporated into undergraduate biology curriculum through lab experiences to immerse students in modern technology that is rapidly changing the landscape of science, medicine and agriculture. We developed and implemented an educational module that introduces students to CRISPR-cas technology in a Genetic course and an Advanced Genetics course. Our primary teaching objective was to immerse students in the design, strategy, conceptual modeling, and application of CRISPR-cas technology using the current research claim of the modification of the CCR5 gene in twin girls. This also allowed us to engage students in an open conversation about the bioethical implications of heritable germline and non-heritable somatic genomic editing. We assessed student-learning outcomes and conclude that this learning module is an effective strategy for teaching undergraduates the fundamentals and application of CRISPR-cas gene editing technology and can be adapted to other genes and diseases that are currently being treated with CRISPR-cas technology.

A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia.

The cilium is a microtubule-based organelle that contains a unique complement of proteins for cell motility and signalling functions. Entry into the ciliary compartment is proposed to be regulated at the base of the cilium. Recent work demonstrated that components of the nuclear import machinery, including the Ran GTPase and importins, regulate ciliary entry. We hypothesized that the ciliary base contains a ciliary pore complex whose molecular nature and selective mechanism are similar to those of the nuclear pore complex. By microinjecting fluorescently labelled dextrans and recombinant proteins of various sizes, we characterize a size-dependent diffusion barrier for the entry of cytoplasmic molecules into primary cilia in mammalian cells. We demonstrate that nucleoporins localize to the base of primary and motile cilia and that microinjection of nucleoporin-function-blocking reagents blocks the ciliary entry of kinesin-2 KIF17 motors. Together, this work demonstrates that the physical and molecular nature of the ciliary pore complex is similar to that of the nuclear pore complex, and further extends functional parallels between nuclear and ciliary import.

Kinesin motors and primary cilia.

Cilia and flagella play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in ciliary assembly and/or function can lead to a range of human diseases, collectively known as the ciliopathies, including polycystic kidney, liver and pancreatic diseases, sterility, obesity, situs inversus, hydrocephalus and retinal degeneration. A basic understanding of how cilia form and function is essential for deciphering ciliopathies and generating therapeutic treatments. The cilium is a unique compartment that contains a distinct complement of protein and lipid. However, the molecular mechanisms by which soluble and membrane protein components are targeted to and trafficked into the cilium are not well understood. Cilia are generated and maintained by IFT (intraflagellar transport) in which IFT cargoes are transported along axonemal microtubules by kinesin and dynein motors. A variety of genetic, biochemical and cell biological approaches has established the heterotrimeric kinesin-2 motor as the 'core' IFT motor, whereas other members of the kinesin-2, kinesin-3 and kinesin-4 families function as 'accessory' motors for the transport of specific cargoes in diverse cell types. Motors of the kinesin-9 and kinesin-13 families play a non-IFT role in regulating ciliary beating or axonemal length, respectively. Entry of kinesin motors and their cargoes into the ciliary compartment requires components of the nuclear import machinery, specifically importin-ß2 (transportin-1) and Ran-GTP (Ran bound to GTP), suggesting that similar mechanisms may regulate entry into the nuclear and ciliary compartments.

Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP.

The biogenesis, maintenance and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. Here, we identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLSs) suggest that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a ciliary-cytoplasmic gradient of the small GTPase Ran, with high levels of GTP-bound Ran (RanGTP) in the cilium. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with the nuclear import protein importin-beta2 in a manner dependent on the CLS and inhibited by RanGTP. We propose that Ran has a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments.