One-step Generation Research Articles

Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.

Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%–58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

Cobalt sulfide/N,S codoped porous carbon core-shell nanocomposites as superior bifunctional electrocatalysts for oxygen reduction and evolution reactions.

Exploring highly-efficient and low-cost bifunctional electrocatalysts for both oxygen reduction reaction (ORR) and oxygen evolution reactions (OER) in the renewable energy area has gained momentum but still remains a significant challenge. Here we present a simple but efficient method that utilizes ZIF-67 as the precursor and template for the one-step generation of homogeneous dispersed cobalt sulfide/N,S-codoped porous carbon nanocomposites as high-performance electrocatalysts. Due to the favourable molecular-like structural features and uniform dispersed active sites in the precursor, the resulting nanocomposites, possessing a unique core-shell structure, high porosity, homogeneous dispersion of active components together with N and S-doping effects, not only show excellent electrocatalytic activity towards ORR with the high onset potential (around -0.04 V vs.-0.02 V for the benchmark Pt/C catalyst) and four-electron pathway and OER with a small overpotential of 0.47 V for 10 mA cm(-2) current density, but also exhibit superior stability (92%) to the commercial Pt/C catalyst (74%) in ORR and promising OER stability (80%) with good methanol tolerance. Our findings suggest that the transition metal sulfide-porous carbon nanocomposites derived from the one-step simultaneous sulfurization and carbonization of zeolitic imidazolate frameworks are excellent alternative bifunctional electrocatalysts towards ORR and OER in the next generation of energy storage and conversion technologies.

Conditional Knockouts Generated by Engineered CRISPR-Cas9 Endonuclease Reveal the Roles of Coronin in C. elegans Neural Development

Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research.

An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome Editing in Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) offer a unique platform for elucidating the genes and molecular pathways that underlie complex traits and diseases. To realize this promise, methods for rapid and controllable genetic manipulations are urgently needed. By combining two newly developed gene-editing tools, the TALEN and CRISPR/Cas systems, we have developed a genome-engineering platform in hPSCs, which we named iCRISPR. iCRISPR enabled rapid and highly efficient generation of biallelic knockout hPSCs for loss-of-function studies, as well as homozygous knockin hPSCs with specific nucleotide alterations for precise modeling of disease conditions. We further demonstrate efficient one-step generation of double- and triple-gene knockout hPSC lines, as well as stage-specific inducible gene knockout during hPSC differentiation. Thus the iCRISPR platform is uniquely suited for dissection of complex genetic interactions and pleiotropic gene functions in human disease studies and has the potential to support high-throughput genetic analysis in hPSCs.

Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.

The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTau(P301L)-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.

Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Dual sgRNAs facilitate CRISPR/Cas9‐mediated mouse genome targeting

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a versatile RNA-guided mammalian genome modification system. One-step generation of mouse genome targeting has been achieved by co-microinjection of one-cell stage embryos with Cas9 mRNA and small/single guide (sg)RNA. Many studies have focused on enhancing the efficiency of this system. In the present study, we report that simultaneous use of dual sgRNAs to target an individual gene significantly improved the Cas9-mediated genome targeting with a bi-allelic modification efficiency of up to 78%. We further observed that the target gene modifications were characterized by efficient germline transmission and site-dependent off-target effects, and also that the apolipoprotein E gene knockout-mediated defects in blood biochemical parameters were recapitulated by CRISPR/Cas9-mediated heritable gene modification. Our results provide a dual sgRNAs strategy to facilitate CRISPR/Cas9-mediated mouse genome targeting.

Once Upon a Mine: The Legacy of Uranium on the Navajo Nation

On a low, windswept rise at the southeastern edge of the Navajo Nation, Jackie Bell-Jefferson prepares to move her family from their home for a temporary stay that could last up to seven years. A mound of uranium-laden waste the size of several football fields, covered with a thin veneer of gravel, dominates the view from her front door. After many years of living next to the contamination and a litany of health problems she believes it caused, Bell-Jefferson and several other local families will have to vacate their homes for a third round of cleanup efforts by the U.S. Environmental Protection Agency (EPA). Decades of uranium mining have dotted the landscape across the Navajo Nation with piles of contaminated mine waste. The EPA has mapped 521 abandoned uranium mines on the reservation, ranging from small holes dug by a single prospector into the side of a mesa to large commercial mining operations.1 The Navajo people did not have a word for “radioactivity” when mining outfits looking for vanadium2 and uranium3 began moving onto their land in the 1940s, and they did not understand that radiation could be dangerous. They were not told that the men who worked in the mines were breathing carcinogenic radon gas and showering in radioactive water, nor that the women washing their husbands’ work clothes could spread radionuclides to the rest of the family’s laundry. Bell-Jefferson and her brother Peterson Bell played in and around the mines, splashing and swimming in pools of radioactive water that had been pumped out of the mines and then collected on their property. The contaminated water looked and tasted perfectly clean. Families used it for cooking, drinking, and cleaning. Hogans and corrals were built with mine wastes, as were roads. All that changed on 16 July 1979. Just about a mile and a half from Bell-Jefferson’s home, a dam broke at the United Nuclear Corporation mill, where workers processed ore from the nearby Northeast Church Rock uranium mine. The spill dumped 94 million gallons of mill process effluent and 1,100 tons of tailings—an acidic, radioactive sludge—into a large arroyo that emptied into the Puerco River.4 The Church Rock spill occurred less than four months after the partial meltdown of the Three Mile Island nuclear reactor, and it released three times as much radiation, making it the biggest nuclear spill in U.S. history, yet it received only a tiny fraction of the news coverage.5 Declared a Superfund site in 1983, the heaps of waste around the mill still cause radiation survey instruments to squeal from the invisible uranium atoms that remain active 30 years later.6 “This area used to be my playground,” Bell-Jefferson says. “Now it’s just a huge wound.” For the Bells and other Dine (the term by which many Navajo people refer to themselves), the Church Rock spill was a turning point. When corporate and government officials appeared in the spill’s aftermath and began inquiring into exposure to the slurry and potential health problems, the Navajo people finally learned the truth—far from being harmless, these uranium mines were poisoning people, and researchers say they will continue to do so for decades to come.

One-step generation of knockout pigs by zygote injection of CRISPR/Cas system

The pig is an important livestock for food supply and an ideal model for various human diseases. Efficient and precise genetic engineering in pigs holds great promise in agriculture and biomedicine1. Using currently available approach, generating specific gene modifications in pigs requires two steps. First, site-specific nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used to generate targeted mutations in pig somatic cells. Then the engineered somatic nucleus is used to generate cloned animals using somatic cell nuclear transfer (SCNT) technology2,3. The complex design and generation of ZFNs and TALENs, as well as the technical challenges of SCNT, greatly limit the application of this method.

Rapid target gene validation in complex cancer mouse models using re‐derived embryonic stem cells

Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

The ultimate step towards a tailored engineering of core@shell and core@shell@shell nanoparticles.

Complex core@shell and core@shell@shell nanoparticles are systems that combine the functionalities of the inner core and outer shell materials together with new physico-chemical properties originated by their low (nano) dimensionality. Such nanoparticles are of prime importance in the fast growing field of nanotechnology as building blocks for more sophisticated systems and a plethora of applications. Here, it is shown that although conceptually simple a modified gas aggregation approach allows the one-step generation of well-controlled complex nanoparticles. In particular, it is demonstrated that the atoms of the core and the shell of the nanoparticles can be easily inverted, avoiding intrinsic constraints of chemical methods.

One-step generation of phenotype-expressing triple-knockout mice with heritable mutated alleles by the CRISPR/Cas9 system.

Triple-knockout mice generated by the one-step CRISPR/Cas9 system were examined for the effects of multiple gene modifications on each phenotype and individual gene function. Sixty embryos were transferred, and 9 pups were obtained; all 9 pups had mutations on 3 loci, and 7 pups showed mutations in all-alleles. F0 mice showed knockout phenotypes or no protein expression of target genes simultaneously, and these mutations were normally inherited in the next generation.

One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering

Taking advantage of the multiplexable genome engineering feature of the CRISPR/Cas9 system, we sought to generate different kinds of immunodeficient mouse strains by embryo co-microinjection of Cas9 mRNA and multiple sgRNAs targeting mouse B2m, Il2rg, Prf1, Prkdc, and Rag1. We successfully achieved multiple gene modifications, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with different heritable genetic modifications at once, providing a one-step strategy for generating different immunodeficient mice which represents significant time-, labor-, and money-saving advantages over traditional approaches. Meanwhile, we improved the technology by optimizing the concentration of Cas9 and sgRNAs and designing two adjacent sgRNAs targeting one exon for each gene, which greatly increased the targeting efficiency and bi-allelic mutations.

One-step generation of covered porous PLGA microspheres for controlled delivery and regenerative medicine

One-step generation of covered porous PLGA microspheres for controlled delivery and regenerative medicine

One-Step Generation of Engineered Drug-Laden Poly(lactic-co-glycolic acid) Micropatterned with Teflon Chips for Potential Application in Tendon Restoration

Regulating cellular behaviors such as cellular spatial arrangement and cellular phenotype is critical for managing tissue microstructure and biological function for engineered tissue regeneration. We herein pattern drug-laden poly(lactic-co-glycolic acid) (PLGA) into grooves using novel Teflon stamps (that possess excellent properties of resistance to harsh organic solvents and molecular adsorption) for engineered tendon-repair therapeutics. The drug release and biological properties of melatonin-laden PLGA grooved micropatterns are investigated. The results reveal that fibroblasts cultured on the melatonin-laden PLGA groove micropatterns not only display significant cell alignment that mimics the cell behavior in native tendon, but also promote the secretion of a major extracellular matrix in tendon, type I collagen, indicating great potential for the engineering of functional tendon regeneration.

One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

A generic scaffold for conversion of peptide ligands into homogenous biosensors

Numerous peptide ligands including protease recognition sequences, peptides mediating protein–protein interactions, peptide epitopes of antibodies and mimotopes are available which bind molecules of interest. However, there is currently no facile method for the incorporation of these peptides into homogenous detection systems. We present a generalizable method for the incorporation of such peptides into a novel fusion protein framework comprising an enzyme and its inhibitor. The incorporated peptide functions as an allosteric hinge, linking enzyme to its inhibitor. Upon interaction with its cognate analyte, the peptide mediates dissociation of the inhibitor from the enzyme, and facilitates one-step signal generation. Likewise, cleavage of the peptide by a specific protease also causes enzyme-inhibitor dissociation, leading to signal generation. Using the β–lactamase Tem1 and its inhibitor protein as a model scaffold, we show both specific and sensitive (between low nanomolar and mid-picomolar) colorimetric detection of proteases and antibodies within minutes in a homogenous one-step reaction visible to the naked eye. The same scaffold affords in vivo detection of antibody binding and protease function by linking activity to a selectable phenotype in E. coli.

One-step generation of multiatom Greenberger–Horne–Zeilinger states in separate cavities via adiabatic passage

We propose a scheme to deterministically generate Greenberger-Horne-Zeilinger states of $N\geq 3$ atoms trapped in spatially separated cavities connected by optical fibers. The scheme is based on the technique of fractional stimulated Raman adiabatic passage which is one-step in the sense that one needs just wait for the desired entangled state to be generated in the stationary regime. The parametrized shapes of the Rabi frequencies of the classical fields that drive the two end atoms are chosen appropriately to realize the scheme. We also show numerically that the proposed scheme is insensitive to the fluctuations of the pulses' parameters and, at the same time, robust against decoherence caused by the dissipation due to fiber decay. Moreover, a relatively high fidelity can be obtained even in the presence of cavity decay and atomic spontaneous emission.

One-step microfluidic generation of pre-hatching embryo-like core-shell microcapsules for miniaturized 3D culture of pluripotent stem cells.

A novel core-shell microcapsule system is developed in this study to mimic the miniaturized 3D architecture of pre-hatching embryos with an aqueous liquid-like core of embryonic cells and a hydrogel-shell of zona pellucida. This is done by microfabricating a non-planar microfluidic flow-focusing device that enables one-step generation of microcapsules with an alginate hydrogel shell and an aqueous liquid core of cells from two aqueous fluids. Mouse embryonic stem (ES) cells encapsulated in the liquid core are found to survive well (>92%). Moreover, ~20 ES cells in the core can proliferate to form a single ES cell aggregate in each microcapsule within 7 days while at least a few hundred cells are usually needed by the commonly used hanging-drop method to form an embryoid body (EB) in each hanging drop. Quantitative RT-PCR analyses show significantly higher expression of pluripotency marker genes in the 3D aggregated ES cells compared to the cells under 2D culture. The aggregated ES cells can be efficiently differentiated into beating cardiomyocytes using a small molecule (cardiogenol C) without complex combination of multiple growth factors. Taken together, the novel 3D microfluidic and pre-hatching embryo-like microcapsule systems are of importance to facilitate in vitro culture of pluripotent stem cells for their ever-increasing use in modern cell-based medicine.