通过 sgRNA-Cas9 RNPs 在. 中进行精确高效的基因组编辑的流水线。
A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in .
机构信息
Department of Molecular Biosciences, Northwestern University , Evanston, IL, USA.
NSF-Simons Center for Quantitative Biology, Northwestern University , Evanston, IL, USA.
出版信息
Fly (Austin). 2020 Mar-Dec;14(1-4):34-48. doi: 10.1080/19336934.2020.1832416. Epub 2020 Oct 21.
Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of as well as to other species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against . This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.
通过同源定向修复（HDR）进行基因组编辑使得对基因序列进行精确和有目的的修饰成为可能。CRISPR/Cas9 介导的 HDR 是实现这一目标的最简单方法。然而，仍然存在技术挑战，需要提高效率并拓宽其适用性，使其不仅适用于任何遗传背景的 ，还适用于其他 物种。为了解决这些问题，我们开发了一种两阶段的标记辅助策略，在该策略中，胚胎被注射 RNP 并使用 T7EI 进行预筛选。我们使用与重组 Cas9 蛋白结合的 sgRNA 来测定每个 sgRNA 的基因组切割效率。然后，我们使用能够有效切割靶基因的 sgRNA 进行 HDR，并应用一种转化标记，该标记针对 产生 RNAi。这允许基于眼睛形态而不是颜色进行筛选。这些新工具可用于在感兴趣的区域进行单个改变或一系列等位基因替换，或创建其他遗传工具，如平衡染色体。
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