Housden Benjamin E, Perrimon Norbert
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):2016/9/pdb.prot090787. doi: 10.1101/pdb.prot090787.
The generation of precise alterations to the genome using CRISPR requires the combination of CRISPR and a donor construct containing homology to the target site. A double-strand break is first generated at the target locus using CRISPR. It is then repaired using the endogenous homologous recombination (HR) pathway. When a donor construct is provided, it can be used as a template for HR repair and can therefore be exploited to introduce alterations in the genomic sequence with single base-pair precision. Here we describe a protocol for the generation of donor constructs using Golden Gate assembly and discuss some key considerations for donor construct design for use in Drosophila.
使用CRISPR对基因组进行精确改造需要将CRISPR与含有与靶位点同源性的供体构建体相结合。首先使用CRISPR在靶位点产生双链断裂。然后使用内源性同源重组（HR）途径进行修复。当提供供体构建体时，它可以用作HR修复的模板，因此可以用于以单碱基对精度引入基因组序列的改变。在这里，我们描述了一种使用金门组装法生成供体构建体的方案，并讨论了用于果蝇的供体构建体设计的一些关键考虑因素。
