Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus
Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus
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DOI：
10.1007/s00427-020-00651-y
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发表时间：
2020-02-06
影响因子：
2.4
通讯作者：
Okumura, Misako
中科院分区：
文献类型：
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作者：
Nakayama, Ken-ichi;Ishita, Yuuki;Okumura, Misako
CRISPR/Cas9基因组编辑方法用于揭示包括线虫在内的许多物种中基因的功能和生物学过程的分子机制。在进化生物学中，太平洋棱纹线虫是一个卫星模型，并已被用来理解有趣的现象，如表型可塑性和自我识别。在太平洋对虾中，CRISPR/Cas9介导的突变是通过将指导RNA（gRNA）和Cas9蛋白显微注射到性腺中来诱导的。然而，由于缺乏视觉标记，突变体筛选是费力且耗时的。在这项研究中，我们通过使用太平洋对虾中的显性滚轴标记建立了Co-CRISPR策略。我们发现Ppa-prl-1的杂合突变诱导了滚轴表型，这可以用作注射标记物。在共注射Ppa-prl-1 gRNA、靶gRNA和Cas9蛋白后，使用异源双链体迁移率测定和DNA测序检查滚子后代及其同胞。我们发现，一些辊和非辊的兄弟姐妹在靶位点突变。我们使用不同浓度的Cas9，发现更高浓度的Cas9不会增加基因组编辑事件。Co-CRISPR策略促进了对基因组编辑事件的筛选，并将促进太平洋对虾新基因组编辑方法的开发。
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.