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Cold Spring Harbor Protocols, 9(2016), p. pdb.prot090787

DOI: 10.1101/pdb.prot090787

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Design and Generation of Donor Constructs for Genome Engineering in Drosophila

Journal article published in 2016 by Benjamin E. Housden, Norbert Perrimon ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Data provided by SHERPA/RoMEO

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.