Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 50, Pages 19821-19826Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0810475105
Keywords
coilin; DNA ligase IV; DNA repair; PAS kinase; targeted mutagenesis
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Funding
- National Institutes of Health [R01 GM078571, R01 GM33397]
- University of Utah Cancer Center
- Medical Research Council [MC_U137788471] Funding Source: researchfish
- MRC [MC_U137788471] Funding Source: UKRI
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We report very high gene targeting frequencies in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild type for DNA repair, the products are dominated by NHEJ mutations. In recipients deficient in the NHEJ component, DNA ligase IV, the majority of products arise by HR with a coinjected donor DNA, with no loss of overall efficiency in target modification. We describe the application of the ZFN injection procedure to mutagenesis by NHEJ of 2 new genes in Drosophila melanogaster: coil and pask. Pairs of novel ZFNs designed for targets within those genes led to the production of null mutations at each locus. The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations.
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