Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls.

Lucas D Sanor,Craig M Crews,Grant Parker Flowers

doi:10.7554/elife.48511

Abstract

Axolotls and other salamanders can regenerate entire limbs after amputation as adults, and much recent effort has sought to identify the molecular programs controlling this process. While targeted mutagenesis approaches like CRISPR/Cas9 now permit gene-level investigation of these mechanisms, genetic screening in the axolotl requires an extensive commitment of time and space. Previously, we quantified CRISPR/Cas9-generated mutations in the limbs of mosaic mutant axolotls before and after regeneration and found that the regenerated limb is a highfidelity replicate of the original limb (Flowers et al. 2017). Here, we circumvent aforementioned genetic screening limitations and present methods for a multiplex CRISPR/Cas9 haploid screen in chimeric axolotls (MuCHaChA), which is a novel platform for haploid genetic screening in animals to identify genes essential for limb regeneration.

Highlights

Salamanders are the only vertebrates known to regenerate complete limbs as adults
Our data suggests that cells lacking the limb blastema-enriched genes, fetub and catalase, have a reduced capacity to contribute to the regenerating limb
Catalase is an enzyme that plays a conserved role in protecting cells from oxidative damage by catalyzing the decomposition of hydrogen peroxide, a reactive oxygen species (ROS)

Summary

Introduction

The axolotl, a species of salamander, can regenerate limbs, tails, and gills without scarring. Regeneration of these complex structures occurs through the formation of a blastema, a mass of proliferating dedifferentiated cells and pre-existing progenitor and stem cells (Currie et al, 2016; Kragl et al, 2009; Sandoval-Guzman et al, 2014). Nearcomplete knockout F0 animals can be generated, they appear to be universally mosaic, harboring a variety of mutant alleles (Flowers et al, 2014). Such embryonically generated mutations both perturb the function of the targeted gene and uniquely label affected cell lineages with a traceable genetic barcode. These data indicate that limb regeneration is a high-fidelity process in which the contributions of small cell populations to the original limb are replicated in the regenerated limb (Figure 1C,D)

Methods

Results

Conclusion