CRISPR Correction of the GBA Mutation in Human-Induced Pluripotent Stem Cells Restores Normal Function to Gaucher Macrophages and Increases Their Susceptibility to Mycobacterium tuberculosis.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the ß-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells.

The ability to target methylation to specific genomic sites would further the study of DNA methylation's biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5'-YGGCCR-3', we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.

Teaching synthetic biology, bioinformatics and engineering to undergraduates: the interdisciplinary Build-a-Genome course.

A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into approximately 750-bp fragments. Once trained in assembly of such DNA "building blocks" by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular "lab meeting" sessions help prepare them for future roles in laboratory science.

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.

Custom-designed molecular scissors for site-specific manipulation of the plant and mammalian genomes.

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity.

The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency.

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells.

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting--the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for 'directed mutagenesis' and targeted 'gene editing' of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

Recent advances in the use of ZFN-mediated gene editing for human gene therapy.

Targeted genome editing with programmable nucleases has revolutionized biomedical research. The ability to make site-specific modifications to the human genome, has invoked a paradigm shift in gene therapy. Using gene editing technologies, the sequence in the human genome can now be precisely engineered to achieve a therapeutic effect. Zinc finger nucleases (ZFNs) were the first programmable nucleases designed to target and cleave custom sites. This article summarizes the advances in the use of ZFN-mediated gene editing for human gene therapy and discusses the challenges associated with translating this gene editing technology into clinical use.

Design of a synthetic yeast genome.

We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross." Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions.

We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT). Binding of the zinc finger domain to the target binding site results in a 10-fold increase in chloramphenicol resistance with the CAT reporter and an 8- to 22-fold increase in total cell fluorescence with the GFP reporter. The CAT reporter allows for sequence specific zinc fingers to be isolated in a single selection step whereas the GFP reporter enables quantitative evaluation of libraries using flow cytometry and theoretically allows for both negative and positive selection. Both systems have been used to select for zinc fingers that have affinity for the motif 5'-GGGGCAGAA-3' from a library of approximately 2 x 10(5) variants. The systems have been engineered to report on zinc finger-DNA binding with dissociation constants less than about 1 microM in order to be most applicable for evaluating binding specificity in an in vivo setting.

Origins of Programmable Nucleases for Genome Engineering.

Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut at off-target sites with mutagenic consequences. Therefore, issues such as efficacy, specificity and delivery are likely to drive selection of reagents for particular purposes. Human therapeutic applications of these technologies will ultimately depend on risk versus benefit analysis and informed consent.

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.

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 Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for ß-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 ß- globin alleles (ßS/ßS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated ßS to the wild-type ßA 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 ßS/ßA hiPSCs, which express the wild-type (ßA) transcript to 30-40% level as compared to uncorrected (ßS/ßS) 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.

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.

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.

The Build-a-Genome course.

Build-a-Genome is an intensive laboratory course at Johns Hopkins University that introduces undergraduates to the burgeoning field of synthetic biology. In addition to lectures that provide a comprehensive overview of the field, the course contains a unique laboratory component in which the students contribute to an actual, ongoing project to construct the first synthetic eukaryotic cell, a yeast cell composed of man-made parts. In doing so, the students acquire basic molecular biology skills and gain a truly "graduate student-like experience" in which they take ownership of their projects, troubleshoot their own experiments, present at frequent laboratory meetings, and are given 24-h access to the laboratory, albeit with all the guidance they will need to complete their projects during the semester. In this chapter, we describe the organization of the course and provide advice for anyone interested in starting a similar course at their own institution.