CRISPR/Cas9 knockin methodology for the sea urchin embryo.
CRISPR/Cas9 knockin methodology for the sea urchin embryo.
复制标题
海胆胚胎的 CRISPR/Cas9 敲入方法。
DOI：
10.1002/mrd.23672
复制
发表时间：
2023
影响因子：
2.5
通讯作者：
Wessel,Gary
中科院分区：
文献类型：
--
作者：
Oulhen,Nathalie;Morita,Shumpei;Warner,JacobF;Wessel,Gary
CRISPR技术彻底改变了生物学研究领域，使动物迄今为止无法进行基因操作，可以分析特定的基因功能。基于海胆中靶向基因突变（CRISPR敲除）的证明（Fleming等人，2021; Lin等人，2019; Lin & Su，2016; Liu等人，2019; Vyas等人，2022），研究人员现在可以将外源DNA插入基因组中的特定位置（CRISPR敲入）。这种Cas9介导的敲入将揭示基因表达和功能的位点。通过明智地选择外源编码的标签，例如荧光报告，研究者可以在活胚胎的整个发育过程中跟踪特定的基因活性和细胞谱系。该标签也可以用于蛋白质下拉，而不需要针对靶蛋白的抗体。在这里，我们描述了一种在海胆中基于CRISPR的敲入DNA的程序。海胆幼虫产生棘色素，其需要几种基因功能，包括酶聚酮合酶1（PKS）（Barsi et al. 2015; Calestani等人，2003; Calestani & Wessel，2018; Perillo等人，2020; Wessel等人，2020年）。Sp PKS 1表达限于动物Veg 2谱系的约50个细胞的小群体（Calestani等人，2003; Barsi等人，2015年）。我们意识到，使用PKS 1来评估CRISPR敲入成功是非常严格的，因为插入必须发生在该小谱系内，并且由该谱系的更小群体表达。通过CRISPR敲除编码PKS 1的基因突变导致白化病幼虫，这是一种易于用简单明场显微镜评估的表型（Oulhen & Wessel，2016 a）。先前显示单个gRNA通过Cas9活性使PKS 1突变，在来自S. purpuratus和Hemicentrotus pulcherrimus（Liu等人，2019; Oulhen等人，2022; Oulhen & Wessel，2016 a）。我们利用这种高效的gRNA来测试和优化海胆中的Cas9介导的方法。
CRISPR technology has revolutionized the biological research world, making animals heretofore recalcitrant to genetic manipulation, accessible to analysis of specific gene functions. Building upon the demonstration of targeted gene mutations in the sea urchin (CRISPR knock-out)(Fleming et al., 2021; Lin et al., 2019; Lin & Su, 2016; Liu et al., 2019; Vyas et al., 2022), investigators may now be able to insert exogenous DNA into specific locations in the genome (CRISPR knock-in). Such Cas9-mediated knock-ins will reveal sites of gene expression, and function. By judicious selection of exogenously encoded tags eg a fluorescent reporter, an investigator may then follow specific gene activities and cell lineages throughout development in live embryos. This tag can also be used for protein pull-down without requiring an antibody for the targeted protein. Here we describe a procedure for CRISPR-based knock-in DNA in the sea urchin Strongylocentrotus purpuratus.Sea urchin larvae produce echinochrome pigments that require several gene functions including the enzyme polyketide synthase 1 (PKS)(Barsi et al., 2015; Calestani et al., 2003; Calestani & Wessel, 2018; Perillo et al., 2020; Wessel et al., 2020). Sp PKS1 expression is restricted to a small population of~ 50 cells of the Veg2 lineage of the animal (Calestani et al., 2003; Barsi et al., 2015). We realized that using PKS1 to evaluate CRISPR knock-in success was highly stringent since the insertion must occur within that small lineage, and be expressed by yet a smaller population of the lineage. Mutations of the gene encoding PKS1 by CRISPR knock-out resulted in albino larvae, an easy phenotype to assess with simple brightfield microscopy (Oulhen & Wessel, 2016a). A single gRNA was previously shown to mutate PKS1 by Cas9 activity, nearly 100% of the time in embryos from S. purpuratus and Hemicentrotus pulcherrimus (Liu et al., 2019; Oulhen et al., 2022; Oulhen & Wessel, 2016a). We took advantage of this highly efficient gRNA to test and to optimize Cas9-mediated methodology in the sea urchin Strongylocentrotus purpuratus.