Design and Generation of Donor Constructs for Genome Engineering in Drosophila
- 1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;
- 2Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115
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.
Footnotes
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↵3 Correspondence: bhousden{at}genetics.med.harvard.edu; perrimon{at}receptor.med.harvard.edu
- © 2016 Cold Spring Harbor Laboratory Press
