TALEN-mediated gene editing in terrestrial and aquatic species.

TALENs are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease.   TALEN induced double strand breaks are useful for gene inactivation via repair by non-homologous end joining (NHEJ) or to stimulate homologous recombination with a user specified template to create custom designed disease alleles.  We found that over 50% of our improved GoldyTALEN displayed activity in livestock fibroblasts with typical percentage of indel positive chromosomes ranging from 20-45%.  Isolation of individual clones with mono- and bi-allelic modification to the target loci was extremely efficient (up to 84% and 24% respectively) and could be accomplished without the aid of selection.   In addition, co-transfection of TALENs with a homologous repair template enabled precise insertion of a novel restriction site in up to 60% of treated cells, 1/3 of which are modified at both alleles.  TALEN modified fibroblasts were suitable for cloning and we’ve generated 17 Ossabaw pigs with mutations in Low-Density Lipoprotein Receptor (LDLR) to model familial hypercholesterolemia.  The GoldyTALEN platform was also useful in vivo gene editing in zebrafish and tilapia.  Efficacy of TALEN mediated NHEJ in zebrafish was near 100% for select loci resulting in bi-allelic knockout and phenotypes consistent with null alleles.  In addition, TALENs co-injected with homologous oligonucleotides induced homology dependent repair in zebrafish allowing seamless introduction of custom alleles at defined locations.  These data suggest GoldyTALENs will be suitable platform for gene editing in commercially relevant aquaculture species, and potential applications will be discussed.  With demonstrated activity in cattle, pigs, chicken and fish, the GoldyTALENs represent a versatile platform for gene editing the most prominent meat producing animals.