title: Design and Generation of Donor Constructs for Genome Engineering in Drosophila.
abstract: 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.
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;
authors: Benjamin E
authors: BE
authors: Housden
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
authors: Norbert
authors: N
authors: Perrimon
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004330:Drosophila / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems