Design and Generation of Donor Constructs for Genome Engineering in Drosophila

Cold Spring Harb Protoc. 2016 Sep 1;2016(9). doi: 10.1101/pdb.prot090787.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • DNA Breaks, Double-Stranded
  • DNA Repair
  • Drosophila / genetics*
  • Gene Editing / methods*
  • Gene Expression*
  • Genetic Vectors*
  • Plasmids
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Recombination, Genetic

Substances

  • RNA, Guide, CRISPR-Cas Systems