State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, the Chinese Academy of Sciences, Beijing 100101, China.
Biol Open. 2014 Apr 15;3(4):271-80. doi: 10.1242/bio.20147682.
Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the "ends-in" recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.
修改许多生物体的基因组变得像在试管中操作 DNA 一样容易，这得益于两种最近开发的技术，它们基于可定制的 DNA 结合蛋白 TALEN 或 CRISPR/Cas9 系统。在这里，我们描述了一系列高效的应用，这些应用结合了各种同源供体 DNA 质粒，用于操纵果蝇基因组：（1）精确产生基因组缺失；（2）以单核苷酸分辨率进行基因组替换 DNA 片段；（3）生成精确插入以标记靶蛋白，用于追踪其内源表达。为了更方便的基因组操作，我们通过同源重组敲入一个白色标记建立了一个易于筛选的平台。此外，我们提供了一种策略来去除“末端入”重组过程中产生的不需要的重复。我们的结果还表明，TALEN 和 CRISPR/Cas9 在介导 HDR（同源定向修复）介导的基因组修饰方面效率相当；TALEN 或 CRISPR/Cas9 系统都可以有效地介导高达 5 kb 的果蝇体内 DNA 片段的替换，为果蝇基因组的功能注释提供了理想的遗传工具。
