使用 ssODN 和 Co-CRISPR 方法在体内高效引入点突变。,Biological Procedures Online

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使用 ssODN 和 Co-CRISPR 方法在体内高效引入点突变。
Biological Procedures Online ( IF 3.7 ) Pub Date : 2020-07-14 , DOI: 10.1186/s12575-020-00123-7
Tgst Levi ₁ , Anna Sloutskin ₂ , Rachel Kalifa ₁ , Tamar Juven-Gershon ₂ , Offer Gerlitz ₁

Affiliation

点突变的产生是评估特定核苷酸或氨基酸在调节或功能环境中的作用的主要工具。然而，在体内检查这些突变需要生成仅在内源基因组位点携带相关点突变的动物，这在技术上具有挑战性。基于CRISPR-Cas9的基因组编辑极大地促进了这种转基因动物的产生；然而，大多数描述的方法使用双链 DNA (dsDNA) 作为供体模板。dsDNA 质粒经常经历不希望的整合事件进入目标基因组位点。使用单链寡脱氧核苷酸 (ssODN) 作为供体模板可防止这种并发症，因此是引入点突变的首选，以及短序列，如蛋白质标签。我们成功地应用了基于 CRISPR 的白色共转化策略和 ssODN 模板，而不是最初描述的 dsDNA 质粒，以创建转基因黑腹果蝇菌株。我们使用该技术轻松地在两条不同的染色体中引入点突变。使用生成的果蝇，我们能够证明各自突变的体内重要性。对于 Nucleoporin107 (Nup107) 基因，1090G > A 突变被证实影响卵巢发育，而对于 tinman (tin) 基因，下游核心启动子元件 (DPE) 的调节作用在发育中的黑腹果蝇胚胎中得到证实。所描述的方法有助于在两个不同的染色体中成功产生点突变，由两个不同的实验室。确定了与新生成的基因型相关的不同表型，从而说明了研究特定核苷酸的体内作用的重要性。此外，还提供了详细的指导方针、建议和交叉计划，以支持科学界产生更多的转基因动物。

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Efficient In Vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR Approach.

The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape. However, examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. The CRISPR-Cas9 based genome editing greatly facilitates the generation of such genetically modified animals; however, most of the described methods use double-strand DNA (dsDNA) as the donor template. The dsDNA plasmids frequently undergo undesired integration events into the targeted genomic locus. The use of a single-strand oligodeoxynucleotide (ssODN) as the donor template prevents this complication and is therefore the preferred choice for introducing point mutations, as well as short sequences such as protein tags. We successfully applied the CRISPR-based white co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified Drosophila melanogaster strains. We used the technique to easily introduce point mutations in two distinct chromosomes. Using the generated flies, we were able to demonstrate the in vivo importance of the respective mutations. For the Nucleoporin107 (Nup107) gene, the 1090G > A mutation was confirmed to affect ovarian development, while for the tinman (tin) gene, the regulatory role of the downstream core promoter element (DPE) was demonstrated within the developing Drosophila melanogaster embryo. The described approach has facilitated the successful generation of point mutations in two different chromosomes, by two different labs. Distinct phenotypes associated with the newly-generated genotype were identified, thus exemplifying the importance of investigating the in vivo role of specific nucleotides. In addition, detailed guidelines, recommendations and crossing schemes are provided in order to support the generation of additional genetically modified animals by the scientific community.

更新日期：2020-07-24

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