Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States.
Elife. 2019 Nov 1;8:e51539. doi: 10.7554/eLife.51539.
We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.
我们之前报道了一种 CRISPR 介导的基因内含子敲入策略，生成了一个用于多种用途的转基因文库（Lee 等人，2018a）。该方法依赖于具有约 1 kb 同源臂的双链 DNA（dsDNA）供体。在这里，我们描述了三种在果蝇中编辑基因的新方法。我们使用 PCR 生成单链 DNA（ssDNA）供体，并在整合盒的每一侧添加 100 个核苷酸的同源性，然后用酶去除一条链。使用这种方法，我们生成了 GFP 标记的蛋白质，标记 S2 细胞中的细胞器。然后，我们描述了两种 dsDNA 方法，使用便宜的合成供体，其两侧带有 100 个核苷酸的同源臂和 gRNA 靶位点克隆到质粒中。注射后，Cas9 从质粒中释放出 1 到 5 kb 的供体 DNA。该盒在体内高效且精确地整合。该方法快速、廉价且可扩展。
