An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.
An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.
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DOI：
10.7554/elife.76077
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发表时间：
2022-06-20
期刊：
影响因子：
7.7
通讯作者：
Bellen, Hugo J.
中科院分区：
文献类型：
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作者：
Kanca, Oguz;Zirin, Jonathan;Hu, Yanhui;Tepe, Burak;Dutta, Debdeep;Lin, Wen-Wen;Ma, Liwen;Ge, Ming;Zuo, Zhongyuan;Liu, Lu-Ping;Levis, Robert W.;Perrimon, Norbert;Bellen, Hugo J.
之前，我们描述了大量的果蝇菌株，每个菌株都携带一个人工外显子，其中包含一个T2AGAL4盒，该盒基于crispr介导的同源重组插入靶基因的内含子中。这些等位基因允许许多应用，并已被证明是非常有用的。最初，基于同源重组的供体构建体具有较长的同源臂（>500 bps），以促进大构建体（>5 kb）的精确整合。最近，我们发现供体结构的体内线性化可以使用短同源臂（100-200 bps）在内含子中插入大的人工外显子。较短的同源臂使得商业合成同源供体成为可能，并且减少了供体构建的克隆步骤。不幸的是，大约58%的果蝇基因缺乏合适的编码内含子来整合所有注释同种异构体中的人工外显子。在这里，我们报道了一组新的构建体的发展，这些构建体允许用KozakGAL4盒式取代缺乏合适内含子的基因的编码区域，从而产生一个敲除/敲入等位基因，该等位基因表达GAL4与靶基因相似。我们还开发了自定义载体主干，以进一步促进和改进转基因。在含有目的基因sgRNA的定制质粒骨架中合成同源供体构建物，避免了注射单独的sgRNA质粒，显著提高了转基因效率。这些升级将使几乎所有的苍蝇基因，无论外显子-内含子结构，以70-80%的成功率靶向。
Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100–200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon–intron structure, with a 70–80% success rate.