Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases.
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.
Beumer
Kelly J
KJ
Department of Biochemistry, University of Utah School of Medicine, 15 North Medical Drive East, Salt Lake City, UT 84112-5650, USA.
Trautman
Jonathan K
JK
Bozas
Ana
A
Liu
Ji-Long
JL
Rutter
Jared
J
Gall
Joseph G
JG
Carroll
Dana
D
D000818:Animals; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D004625:Embryo, Nonmammalian; D004720:Endonucleases / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D008845:Microinjections; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics*; D011995:Recombination, Genetic; D016335:Zinc Fingers / Q000235:genetics*
D012333:RNA, Messenger; D004720:Endonucleases; D011088:DNA Ligases; D000072481:DNA Ligase ATP