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.

中文翻译：

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在太平洋线虫Pristionchus pacificus中使用共注射标记物筛选CRISPR / Cas9诱导的突变。

CRISPR / Cas9基因组编辑方法用于揭示包括线虫在内的许多物种的生物过程背后的基因功能和分子机制。在进化生物学中，线虫Pristionchus pacificus是一种卫星模型，已被用于理解有趣的现象，例如表型可塑性和自我识别。在P. pacificus中，通过向性腺微注射向导RNA（gRNA）和Cas9蛋白来诱导CRISPR / Cas9介导的突变。然而，由于缺乏视觉标记，突变体筛选费力且费时。在这项研究中，我们通过在太平洋假单胞菌中使用显性滚筒标记建立了Co-CRISPR策略。我们发现Ppa-prl-1中的杂合突变诱导的滚子表型，可以用作注射标记。共注射Ppa-pr1-1 gRNA，靶标gRNA和Cas9蛋白后，使用异源双链迁移率测定法和DNA测序检查了辊子后代及其兄弟姐妹。我们发现一些轮生和非轮生兄弟姐妹在目标位点具有突变。我们使用了不同的Cas9浓度，发现较高的Cas9浓度不会增加基因组编辑事件。协同CRISPR策略促进了基因组编辑事件的筛选，并将促进太平洋假单胞菌中新的基因组编辑方法的开发。