Sivaprakash Ramalingam

Total Synthesis of a Functional Designer Eukaryotic Chromosome

Designer Chromosome One of the ultimate aims of synthetic biology is to build designer organisms from the ground up. Rapid advances in DNA synthesis has allowed the assembly of complete bacterial genomes. Eukaryotic organisms, with their generally much larger and more complex genomes, present an additional challenge to synthetic biologists. Annaluru et al. (p. 55, published online 27 March) designed a synthetic eukaryotic chromosome based on yeast chromosome III. The designer chromosome, shorn of destabilizing transfer RNA genes and transposons, is ∼14% smaller than its wild-type template and is fully functional with every gene tagged for easy removal. A synthetic version of yeast chromosome III with every gene tagged can substitute for the original. Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

Creating Designed Zinc Finger Nucleases with Minimal Cytotoxicity

Zinc-finger nucleases (ZFNs) have emerged as powerful tools for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination with investigator-provided donor DNA or induce gene mutations at the site of cleavage in the absence of a donor by nonhomologous end joining both in plant cells and in mammalian cells, including human cells. ZFNs are formed by fusing zinc-finger proteins to the nonspecific cleavage domain of the FokI restriction enzyme. ZFN-mediated gene targeting yields high gene modification efficiencies (>10%) in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs. The mechanism of DSB by ZFNs requires (1) two ZFN monomers to bind to their adjacent cognate sites on DNA and (2) the FokI nuclease domains to dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs fused to wild-type FokI cleavage domains, homodimers may also form; this could limit the efficacy and safety of ZFNs by inducing off-target cleavage. In this article, we report further refinements to obligate heterodimer variants of the FokI cleavage domain for the creation of custom ZFNs with minimal cellular toxicity. The efficacy and efficiency of the reengineered obligate heterodimer variants of the FokI cleavage domain were tested using the green fluorescent protein gene targeting reporter system. The three-finger and four-finger zinc-finger protein fusions to the REL_DKK pair among the newly generated FokI nuclease domain variants appear to eliminate or greatly reduce the toxicity of designer ZFNs to human cells.

Targeted manipulation of mammalian genomes using designed zinc finger nucleases.

Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.

A CRISPR way to engineer the human genome

RNA-guided genome engineering based on the type II prokaryotic CRISPR/Cas system provides an efficient and versatile method for targeted manipulation of mammalian genomes.

TALEN-Mediated Generation and Genetic Correction of Disease-Specific Human Induced Pluripotent Stem Cells

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gauchers disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Generation and Genetic Engineering of Human Induced Pluripotent Stem Cells Using Designed Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

Rewriting the blueprint of life by synthetic genomics and genome engineering

Advances in DNA synthesis and assembly methods over the past decade have made it possible to construct genome-size fragments from oligonucleotides. Early work focused on synthesis of small viral genomes, followed by hierarchical synthesis of wild-type bacterial genomes and subsequently on transplantation of synthesized bacterial genomes into closely related recipient strains. More recently, a synthetic designer version of yeast Saccharomyces cerevisiae chromosome III has been generated, with numerous changes from the wild-type sequence without having an impact on cell fitness and phenotype, suggesting plasticity of the yeast genome. A project to generate the first synthetic yeast genome - the Sc2.0 Project - is currently underway.

Pharmacological and molecular approaches for the treatment of β-hemoglobin disorders.

β‐hemoglobin disorders, such as β‐thalassemia and sickle cell anemia are among the most prevalent inherited genetic disorders worldwide. These disorders are caused by mutations in the gene encoding hemoglobin‐β (HBB), a vital protein found in red blood cells (RBCs) that carries oxygen from lungs to all parts of the human body. As a consequence, there has been an enduring interest in this field in formulating therapeutic strategies for the treatment of these diseases. Currently, there is no cure available for hemoglobin disorders, although, some patients have been treated with bone marrow transplantation, whose scope is limited because of the difficulty in finding a histocompatible donor and also due to transplant‐associated clinical complications that can arise during the treatment. On account of these constraints, reactivation of fetal hemoglobin (HbF) synthesis holds immense promise and is a viable strategy to alleviate the symptoms of β‐hemoglobin disorders. Development of new genomic tools has led to the identification of important natural genetic modifiers of hemoglobin switching which include BCL11A, KLF1, HBSIL‐MYB, LRF, LSD1, LDB1, histone deacetylases 1 and 2 (HDAC1 and HDAC2). miRNAs are also promising therapeutic targets for development of more effective strategies for the induction of HbF production. Many new small molecule pharmacological inducers of HbF production are already under pre‐clinical and clinical development. Furthermore, recent advancements in gene and cell therapy includes targeted genome editing and iPS cell technologies, both of which utilizes a patients own cells, are emerging as extremely promising approaches for significantly reducing the burden of β‐hemoglobin disorders.