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Abstract：
Here we describe a Drosophila genome engineering technique that can scarlessly modify genomic sequences near any mapped attP attachment site previously integrated by transposon mobilization or gene targeting. This technique combines two highly efficient and robust procedures: phiC31 integrase-mediated site-specific integration and homing endonuclease-mediated resolution of local duplications. In this technique, a donor fragment containing the desired mutation(s) is first integrated into a selected attP site near the target locus by phiC31 integrase-mediated site-specific integration, which creates local duplications consisting of the mutant-containing donor fragment and the wild-type target locus. Next, homing endonuclease-induced double-stranded DNA breaks trigger recombination between the duplications and resolve the target locus to generate scarless mutant alleles. In every step, the desired flies can be easily identified by patterns of dominant markers, so no large-scale screens are needed. This technique is highly efficient and can be used to generate scarless point mutations, insertions, and deletions. The availability of large libraries of mapped attP site-containing transposon/CRISPR insertions in Drosophila allows the modification of more than half of the euchromatic Drosophila genome at a high efficiency. As more and more attP-containing insertions are generated and mapped, this technique will be able to modify larger portions of the Drosophila genome. The principles of this technique are applicable to other organisms where modifications to the genome are feasible. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Verifying attP-containing insertions Support Protocol: Extracting genomic DNA Basic Protocol 2: Generating the donor plasmid Basic Protocol 3: Injecting the donor plasmid and establishing transformant stocks Basic Protocol 4: Verifying the transformants Basic Protocol 5: Generating the final scarless alleles Basic Protocol 6: Verifying the final alleles.
摘要：
在这里，我们描述了一种果蝇基因组工程技术，该技术可以毫不费力地修饰先前通过转座子动员或基因靶向整合的任何映射的attP附着位点附近的基因组序列。该技术结合了两种高效且可靠的程序：phiC31整合酶介导的位点特异性整合和归巢核酸内切酶介导的局部重复分辨率。在这项技术中,首先通过phiC31整合酶介导的位点特异性整合将含有所需突变的供体片段整合到靶基因座附近的选定attP位点中，其产生由含有突变体的供体片段和野生型靶基因座组成的局部重复。接下来,归巢核酸内切酶诱导的双链DNA断裂会触发重复之间的重组，并解析目标基因座以产生无疤痕的突变等位基因。每一步,所需的果蝇可以很容易地通过显性标记的模式来识别，所以不需要大屏幕。这种技术非常有效，可以用来产生无疤痕的点突变，插入,和删除。果蝇中映射的包含attP位点的转座子/CRISPR插入的大型文库的可用性允许高效率地修饰超过一半的常色果蝇基因组。随着越来越多的包含attP的插入被生成和映射，这项技术将能够修改果蝇基因组的较大部分。该技术的原理适用于基因组修饰可行的其他生物体。©2023威利期刊有限责任公司。基本方案1:验证含有attP的插入支持方案:提取基因组DNA基本方案2:产生供体质粒基本方案3:注射供体质粒并建立转化体储备基本方案4:验证转化体基本方案5:产生最终无瘢痕等位基因基本方案6:验证最终等位基因。
