Gregory J. Cost

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.

Translating dosage compensation to trisomy 21

Down’s syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down’s syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a ‘chromosome 21 Barr body’. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of ‘chromosome therapy’.

Smc5p Promotes Faithful Chromosome Transmission and DNA Repair in Saccharomyces cerevisiae

Heterodimers of structural maintenance of chromosomes (SMC) proteins form the core of several protein complexes involved in the organization of DNA, including condensation and cohesion of the chromosomes at metaphase. The functions of the complexes with a heterodimer of Smc5p and Smc6p are less clear. To better understand them, we created two S. cerevisiae strains bearing temperature-sensitive alleles of SMC5. When shifted to the restrictive temperature, both mutants lose viability gradually, concomitant with the appearance of nuclear abnormalities and phosphorylation of the Rad53p DNA damage checkpoint protein. Removal of Rad52p or overexpression of the SUMO ligase Mms21p partially suppresses the temperature sensitivity of smc5 strains and increases their survival at the restrictive temperature. At the permissive temperature, smc5-31 but not smc5-33 cells exhibit hypersensitivity to several DNA-damaging agents despite induction of the DNA damage checkpoint. Similarly, smc5-31 but not smc5-33 cells are killed by overexpression of the SUMO ligase-defective Mms21-SAp but not by overexpression of wild-type Mms21p. Both smc5 alleles are synthetically lethal with mms21-SA and exhibit Rad52p-independent chromosome fragmentation and loss at semipermissive temperatures. Our data indicate a critical role for the S. cerevisiae Smc5/6-containing complexes in both DNA repair and chromosome segregation.

Giant proteins that move DNA: bullies of the genomic playground

As genetic material DNA is wonderful, but as a macromolecule it is unruly, voluminous and fragile. Without the action of DNA replicases, topoisomerases, helicases, translocases and recombinases, the genome would collapse into a topologically entangled random coil that would be useless to the cell. We discuss the organization, movement and energetics of these proteins that are crucial to the preservation of a molecule that has such beautiful biological but challenging physical properties.

Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA

This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique. DNA fragments in ligation reactions are capable of combining to produce numerous products. The ELAN method uses judicious choice of restriction enzyme sites coupled with simultaneous digestion and ligation reactions to create just one product, by converting off-pathway products back into substrate. The experimental parameters critical for a successful ELAN reaction are discussed, and the ordered, one-tube assembly of four DNA fragments in the presence of eight restriction enzymes is demonstrated. This technique will be useful to those performing gene construction, DNA computing, biophysics and even standard molecular cloning. Starting with reactant fragments, the protocol takes 4–16 h to produce nanogram to microgram yields, depending on the complexity of the reaction.

Directed assembly of DNA molecules via simultaneous ligation and digestion.

DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of subsequent manipulations (e.g., selection via bacterial transformation) often obviates the need for a high yield of correctly ligated products. However the ability to perform high-yield, preparative-scale DNA ligations would benefit a number of downstream applications ranging from standard molecular cloning to biophysics and DNA computing. We describe here a ligation technique that specifically converts off-pathway ligation products back into substrate. We term this second-chance strategy enzymatic ligation assisted by nucleases (ELAN) and demonstrate the ordered assembly of four DNA fragments via simultaneous ligation and digestion in the presence of eight restriction enzymes. Use of ELAN increased the yield of the desired product by more than 30-fold.

432. Valproic Acid Treatment Enhances Hematopoietic Stem and Progenitor Cell Multipotency Ex Vivo for Enhanced Long-Term Engraftment of Gene-Modified Cells

The promising field of genome editing in hematopoietic stem and progenitor (HSPC) for use in autologous and allogeneic transplantation therapies relies on being able to engraft the edited cells into the bone marrow and to have those engrafted cells produce all the hematopoietic lineages necessary for proper immune and red blood cell function. Depending on the cell source to be used in the editing process, the fraction of CD34+ HSPCs can be quite low - approximately 0.0005%, 0.01%, or 0.1% for mobilized peripheral blood (mPB), bone marrow aspirate (BM), or cord blood, respectively. The fraction of long-term repopulating true stem cells (LT-HSCs) within these CD34+ cell populations, capable of long-term reconstitution of the entire hematopoietic lineage after transplantation, is even lower (<1%). In addition, it has recently been shown that LT-HSCs derived from cord blood can be much less permissive to homology-directed repair (HDR)-driven gene correction which may be essential for some types of therapeutic genome editing.Several groups have discovered small molecules that promote expansion of cord blood-derived CD34+ HSPCs ex vivo while maintaining the stemness of the HSPCs, however these effects have not been reported in HSPCs from mPB or BM. Here we show that the small-molecule epigenetic modifier valproic acid (VPA) improves HDR-mediated targeted integration (TI) in mPB and BM HSPCs. In addition to increasing the fraction of LT-HSCs with TI by up to 100-fold, VPA also dramatically increases the overall number of cells expressing LT-HSC markers (CD34+CD133+CD90+CD49f+ or CD34+CD38-CD45RA-CD90+CD49f+) by up to 500-fold. VPA-treated gene-edited HSCs differentiate normally in vitro and retain consistent levels of TI in both erythroid and myeloid lineages. These results further the development of genome-edited mPB and BM-derived HSPC therapies.