Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York.
Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, New York, New York.
Curr Protoc. 2023 Aug;3(8):e855. doi: 10.1002/cpz1.855.
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.
我们在这里描述了一种可无痕修饰基因组序列的果蝇基因工程技术，该技术可作用于通过转座子转座或基因靶向整合到任何已定位的 attP 附着位点附近的基因组序列。该技术结合了两种高效且强大的程序：phiC31 整合酶介导的位点特异性整合和归巢内切酶介导的局部重复序列的分辨率。在该技术中，首先通过 phiC31 整合酶介导的位点特异性整合将包含所需突变的供体片段整合到靶基因座附近的选定 attP 位点，该整合会产生包含突变供体片段和野生型靶基因座的局部重复序列。接下来，归巢内切酶诱导的双链 DNA 断裂会引发重复序列之间的重组，并解析靶基因座以产生无痕突变等位基因。在每一步，都可以通过显性标记的模式轻松识别所需的蝇种，因此不需要进行大规模筛选。该技术高效，可用于生成无痕点突变、插入和缺失。在果蝇中，大量已定位的含有 attP 位点的转座子/CRISPR 插入文库的可用性使得高效地修饰超过半数的常染色质果蝇基因组成为可能。随着越来越多的含有 attP 的插入物被生成和定位，该技术将能够修饰更大比例的果蝇基因组。该技术的原理适用于其他可对基因组进行修饰的生物体。© 2023 Wiley Periodicals LLC. 基本方案 1：验证含有 attP 的插入物 支持方案：提取基因组 DNA 基本方案 2：生成供体质粒 基本方案 3：注射供体质粒并建立转化体品系 基本方案 4：验证转化体 基本方案 5：生成最终无痕等位基因 基本方案 6：验证最终等位基因。
