Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration

Developmental biology

Kiyokazu Agata^3,4¹  Miyuki Suzuki^1²  Takeshi Inoue^3³  Miki Hirayama^4⁴  Toshinori Hayashi^2⁵ 

[1]Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Kyoto 606-8502, Japan^4

[2]Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan^1

[3]Graduate Course in Life Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan^3

[4]National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan^5

[5]School of Life Sciences, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan^2

关键词: CRISPR-Cas9;    Genome editing;    Newt;    Regeneration;   

DOI  :  10.1016/j.ydbio.2018.09.008

学科分类：生物科学（综合）

来源: Academic Press

【 摘 要 】

Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase, pax6, tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.

【 授权许可】

CC BY