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ID 51068
file
creator
Nakayama, Ken-ichi
Ishita, Yuuki
subject
Pristionchus pacificus
CRISPR/Cas9
Co-injection marker
Microchip electrophoresis
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.
description
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.
journal title
Development Genes and Evolution
volume
Volume 230
issue
Issue 3
start page
257
end page
264
date of issued
2020-02-06
publisher
Springer
issn
0949-944X
1432-041X
publisher doi
pubmed id
language
eng
nii type
Journal Article
HU type
Journal Articles
DCMI type
text
format
application/pdf
text version
author
rights
This is a post-peer-review, pre-copyedit version of an article published in Development Genes and Evolution. The final authenticated version is available online at: https://doi.org/10.1007/s00427-020-00651-y
This is not the published version. Please cite only the published version. この論文は出版社版ではありません。引用の際には出版社版をご確認、ご利用ください。
relation url
department
Graduate School of Integrated Sciences for Life