Development Of Gene Editing Technologies Research Articles

GENE INACTIVATION AND NONMEIOTIC ALLELE INTROGRESSION IN LIVESTOCK SPECIES USING TALENS

TALEN-induced double-strand breaks can be used for gene inactivation via repair by non-homologous end joining (NHEJ) or to stimulate homologous recombination (HR). HR can be used to introduce custom genetic modifications or to introgress naturally occurring alleles. We found that over 65% of custom-designed TALENs displayed activity in pig and cattle fibroblasts, with a typical percentage of indel positive chromosomes ranging from 20 to 45%. Isolation of individual clones with mono- and biallelic modifications to targeted loci was extremely efficient (up to 84 and 24% of clones, respectively) and could be accomplished without the aid of selection. Co-transfection of TALENs with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, with surprising levels of homozygosity. To prove that gene-edited Ossabaw swine cells were suitable for the generation of animals by cloning, we pooled colonies harboring both monoallelic and biallelic TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for chromatin transfer. Pregnancy was established in 7/9 transfers, and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Pigs heterozygous and homozygous for TALEN-induced mutations are being investigated as models of familial hypercholesterolemia (FH). We have additionally targeted the same locus for HR using a specified inactivating mutation. Fibroblasts heterozygous and homozygous for a specific 4-bp insertion into LDLR were created by allele introgression and have been cloned by chromatin transfer, demonstrating that gene editing can be used to create precise, swine knock-ins in a single generation. Allele introgression is also critical to livestock genetics, where crossbreeding has been a staple of breeding programs. Although major effect alleles for enhancing productivity and animal welfare have been discovered, the introgression of low-frequency alleles by traditional breeding is slow and inaccurate, involving recombination across the entire genome. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement in a diversity of livestock breeds. Co-transfection of a TALEN pair with a template containing a specific, naturally occurring allele was effective at the non-meiotic introgression of quantitative traits into the genome of cells from naïve cattle breeds, now being used to create founders by cloning. We will also present progress towards gene conversion by direct injection of livestock embryos. Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in up to 75% of embryos analysed, nearly half of which harbored biallelic modification. We will present alternative strategies for the incorporation of gene editing into livestock genetic improvement programs by either cloning or embryo treatment.

337 NONMEIOTIC INTROGRESSION OF QUANTITATIVE TRAIT NUCLEOTIDES AND CORRECTION OF CONGENITAL MUTATIONS IN LIVESTOCK WITH TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.