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Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus

Abstract

CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus, CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus. We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus.

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Acknowledgments

We would like to thank Dr. Keisuke Nakajima (Institute for Amphibian Biology, Hiroshima University) and Mr. Nobuo Yamaguchi (Natural Science Center for Basic Research and Development, Hiroshima University) for helping with microchip electrophoresis. We also thank all the members of Chihara laboratory for their kind support and Editage (www.editage.jp) for English language editing.

Funding

This work was supported by JSPS KAKENHI Grant Number 18K14716 to MO and 18H05369 to TC. This work was also supported by Tomizawa Jun-ichi & Keiko Fund of Molecular Biology Society of Japan for Young Scientist and AMED under Grant Number JP19gm6310003 to MO, and Toray Science Foundation, The Frontier Development Program for Genome Editing, and Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers JPMXS05S2900002 to TC.

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Correspondence to Misako Okumura.

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Communicated by Ralf Sommer

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Nakayama, K., Ishita, Y., Chihara, T. et al. Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus. Dev Genes Evol (2020). https://doi.org/10.1007/s00427-020-00651-y

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Keywords

  • Pristionchus pacificus
  • CRISPR/Cas9
  • Co-injection marker
  • Microchip electrophoresis