Chris Proudfoot

Efficient TALEN-mediated gene knockout in livestock

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.

Genome edited sheep and cattle.

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

Live pigs produced from genome edited zygotes

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.

Highly efficient targeted chromosome deletions using CRISPR/Cas9 †

The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user-specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment. Predetermined genomic DNA segments ranging from several-hundred base pairs to 1 Mbp can be precisely deleted at frequencies of 1-10%, with no apparent correlation between the size of the deleted fragment and the deletion frequency. The high efficiency of this technique holds promise for large genomic deletions that could be useful in generation of cell and animal models with engineered chromosomes.

Mammalian interspecies substitution of immune modulatory alleles by genome editing

We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research.

Comparison of CRISPR/Cas9 and TALENs on editing an integrated EGFP gene in the genome of HEK293FT cells

BackgroundGenome editors such as CRISPR/Cas9 and TALENs are at the forefront of research into methodologies for targeted modification of the mammalian genome. To date few comparative studies have been carried out to investigate the difference of genome editing characteristics between CRISPR/Cas9 and TALENs. While the CRISPR/Cas9 system has overtaken TALENs as the tool of choice for most research groups working in this field, we hypothesized that there could be certain applications whereby the application of TALENs would have specific benefits. Here we compare CRISPR/Cas9 and TALEN as tools for introducing site-specific editing events at an integrated EGFP gene in the genome of HEK293FT cells.ResultsGuide RNAs and TALEN pairs were designed to target two loci within the EGFP gene. We found that paired Cas9 nucleases induced targeted genomic deletion more efficiently and precisely than two TALEN pairs. However, when concurrently supplied with a plasmid template spanning the two DNA double-strand breaks (DSBs) within EGFP, TALENs stimulated homology directed repair (HDR) more efficiently than CRISPR/Cas9 and caused fewer targeted genomic deletions.ConclusionsOur data suggest that the choice of genome editing tool should be determined by the desired genome editing outcome. Such a rational approach is likely to benefit research outputs for groups working in fields as diverse as modification of cell lines, to animal models for disease studies, or gene therapy strategies.

Genetically engineering milk.

It has been thirty years since the first genetically engineered animal with altered milk composition was reported. During the intervening years, the world population has increased from 5bn to 7bn people. An increasing demand for protein in the human diet has followed this population expansion, putting huge stress on the food supply chain. Many solutions to the grand challenge of food security for all have been proposed and are currently under investigation and study. Amongst these, genetics still has an important role to play, aiming to continually enable the selection of livestock with enhanced traits. Part of the geneticists tool box is the technology of genetic engineering. In this Invited Review, we indicate that this technology has come a long way, we focus on the genetic engineering of dairy animals and we argue that the new strategies for precision breeding demand proper evaluation as to how they could contribute to the essential increases in agricultural productivity our society must achieve.

Comparison of surrogate reporter systems for enrichment of cells with mutations induced by genome editors

Genome editors are powerful tools that allow modification of the nuclear DNA in eukaryotic cells both in vitro and in vivo. In vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their isolation for analysis. Episomal reporters encoding fluorescent proteins can be used for enrichment of modified cells by flow cytometry. Here we compare two surrogate reporters, RGS and SSA, for the enrichment of porcine embryonic fibroblasts containing mutations induced by ZFNs or CRISPR/Cas9. Both systems were effective for enrichment of edited porcine cells with the RGS reporter proving more effective than the SSA reporter. We noted a higher-fold enrichment when editing events were induced by Cas9 compared to those induced by ZFNs, allowing selection at frequencies as high as 70%.

Genome editing for disease resistance in livestock

One of the major burdens on the livestock industry is loss of animals and decrease in production efficiency due to disease. Advances in sequencing technology and genome-editing techniques provide the unique opportunity to generate animals with improved traits. In this review we discuss the techniques currently applied to genetic manipulation of livestock species and the efforts in making animals disease resistant or resilient.

Editing myostatin in livestock animals

Program and Abstracts of the 12th Transgenic Technology Meeting (TT2014) The Assembly Rooms, Edinburgh, Scotland, United Kingdom, 6–8 October 2014 The TT2014 Meeting is hosted by: The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, United Kingdom. The Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, Scotland United Kingdom. The Institute of Genetics and Molecular Medicine, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU United Kingdom. 123 Transgenic Res (2014) 23:827–909 DOI 10.1007/s11248-014-9820-1 Springer International Publishing Switzerland 2014