Screening can be improved by using haploid cells, which harbor a single copy of each gene, and thus require monoallelic inactivation to unveil loss-of-function phenotypes. Negative selection screens are widely used to identify genes essential for cellular processes with CRISPR/Cas ( Shalem et al., 2014 Wang et al., 2014 Yin and Chen, 2017). We anticipated that genetic perturbation of critical blastema-enriched genes would impair mutagenized cells’ ability to participate in the regenerative process. This suggests a shared genetic program across most blastemal cells.

Recent single-cell sequencing of the axolotl limb blastema demonstrated that cell identities converge at a transcriptional level during regeneration ( Gerber et al., 2018). Mutagenized genes essential for regeneration (red) will show a decrease in allele frequency or a complete loss of alleles in the regenerated limb. Mutant alleles of a neutral gene, tyrosinase (blue), are faithfully preserved between original and regenerated limbs. ( D) A hypothetical linear regression plot of the log 2 of reads per ten thousand (RP10K+1) of mutant alleles before and after regeneration. Cell lineages harboring deleterious mutant alleles deleterious (red, far right) are predicted to be reduced in regenerated limbs. Cell lineages mutant for non-essential candidate genes (light blue, dark blue, yellow, purple, red, green) may participate normally in regeneration and therefore contribute to the regenerated limb and original limb in a similar proportion. ( C) Schematic depicting the contribution of mutant cell lineages to the original and regenerated limb. ( B) DNA is extracted from original and fully regenerated haploid limbs of juvenile chimeric axolotls, target sequences are PCR amplified, and these products are subjected to NGS. Chimeric embryos are generated by replacing the limb buds of GFP+ diploid embryos with corresponding tissue from mutagenized haploid donors.

Gynogenetic haploids are generated by in vitro activation of unfertilized eggs with UV-enucleated sperm and subsequently mutagenized using CRISPR/Cas9. ( A) Schematic of haploid-diploid chimera generation. 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). We found that the majority of very low-frequency alleles reoccur in a regenerated limb at a frequency strikingly similar to that of the original limb.

Previously, we measured the fidelity of limb regeneration by using next-generation sequencing (NGS) to quantify the mutant allele frequencies of multiple genomic loci before and after limb regeneration in mosaic mutant axolotls ( Flowers et al., 2017). Such embryonically generated mutations both perturb the function of the targeted gene and uniquely label affected cell lineages with a traceable genetic barcode. Although near-complete knockout F 0 animals can be generated, they appear to be universally mosaic, harboring a variety of mutant alleles ( Flowers et al., 2014). The advent of CRISPR/Cas9 has made the axolotl a genetically tractable organism and the functional interrogation of these genes possible ( Flowers et al., 2014 Fei et al., 2014). Transcriptional profiling of the limb blastema has produced long lists of candidate genes that, to date, remain largely uncharacterized ( Bryant et al., 2017 Campbell et al., 2011 Gerber et al., 2018 Knapp et al., 2013 Leigh et al., 2018 Monaghan et al., 2009 Voss et al., 2015). 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-Guzmán et al., 2014).

The axolotl, a species of salamander, can regenerate limbs, tails, and gills without scarring. Salamanders are the only vertebrates known to regenerate complete limbs as adults.

Chimera axolotl