Gene Targeting with TALENs in Drosophila. Dana Carroll¹, Kelly J. Beumer¹, Jonathan K. Trautman¹, Michelle Christian², Timothy J. Dahlem³, Cathleen Lake⁴, R. Scott Hawley⁴, David J. Grunwald³, Daniel F. Voytas². 1) Dept Biochem, Univ Utah Sch Med, Salt Lake City, UT; 2) Dept Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN; 3) Dept Human Genetics, Univ Utah Sch Med, Salt Lake City, UT; 4) Stowers Institute, Kansas City, MO.

   Gene targeting in Drosophila is getting easier and more reliable, thanks to a new class of targetable cleavage reagents. TALENs employ DNA binding domains from transcription activator-like effectors (TALEs), linked to a nonspecific cleavage domain, to make double-strand breaks (DSBs) at specific sites in chromosomal DNA. Through the action of cellular repair pathways, these targeted breaks lead to localized mutagenesis via nonhomologous end joining and to gene replacement via homologous recombination. Each TALE repeat binds a single base pair in the DNA target, thereby simplifying design in comparison to ZFNs, and there seem to be fewer context effects than with zinc fingers. Using standard embryo injection procedures to introduce the corresponding mRNAs, we have tested the function of TALENs in Drosophila. In direct comparisons, we found TALENs to be frequently (but not always) more effective than our previously described ZFNs. We have successfully knocked out several genes in which null mutations were not previously available. In addition, we have used oligonucleotide donor DNAs, in conjunction with TALEN cleavage, to introduce specific sequence changes at the target locus. TALENs promise to facilitate the manipulation of natural genomic loci in Drosophila and other organisms to a much greater degree than previous targeting reagents.