Efficient TALEN-mediated gene knockout in livestock

Abstract

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono-and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono-and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.Tal-effector nuclease | biotechnology | gene-editing

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the FrogXenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

“…However, both AHR1a -A3 and AHR1b-B1 were homozygous for their respective mutations. We suggest that this outcome involves homologous recombination rather than independent, identical NHEJ events, as observed in other systems (Carlson et al, 2012;Urnov et al, 2005). Responding to initial double-strand break, the NHEJ repair process generates an indel at a cleaved target site.…”

Efficient nonmeiotic allele introgression in livestock using custom endonucleases

“…In particular, TALEN-induced gene disruption has been demonstrated in various species ranging from model organisms (9)(10)(11)(12) to crops (13,14), farm animals (15), and humans (16)(17)(18). However, indels introduced by NHEJ are variable in size and sequence, which make screening for functionally disrupted clones arduous (15) and do not allow for precise alterations.…”

“…In particular, TALEN-induced gene disruption has been demonstrated in various species ranging from model organisms (9)(10)(11)(12) to crops (13,14), farm animals (15), and humans (16)(17)(18). However, indels introduced by NHEJ are variable in size and sequence, which make screening for functionally disrupted clones arduous (15) and do not allow for precise alterations. TALEN-or CRISPR/ cas9-mediated homology-directed repair (HDR) supports the introduction of defined nucleotide changes; however, so far this has only been demonstrated in lower eukaryotic models, including yeast (19), zebrafish (20), and, very recently, mice (21,22).…”

Generation of RAG 1- and 2-deficient rabbits by embryo microinjection of TALENs

“…Unlike ZFNs, TALENs have fewer off-target effects and lower toxicity. Given the success of the TALEN technology in reported species [4][5][6], we attempted to explore the feasibility of producing KO rabbits using this technology. In the current study, we chose recombination activation genes (RAGs), including RAG1 and RAG2, as the first genes of interest.…”