Amplicon sequencing analysis of genome edited Pleurodeles waltl

Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding and gene knockout has hampered laboratory research in newt. Here, we present a simple and efficient gene knockout method using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a suitable species for laboratory research. Surprisingly, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete disruption at three target loci (tyrosinase, pax6, tbx5) in founders. Moreover, we demonstrated the generation of tyr null F1 offspring within a year. Finally, we assessed the function of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence, during limb development and regeneration in newt. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in newt.