Xingxu Huang

Fabrication and electronic transport properties of Bi nanotube arrays

Bi nanotubes embedded in anodic alumina membranes were fabricated by pulsed electrodeposition. Scanning electron microscope, x-ray diffraction, and high-resolution transmission electron microscope analyses revealed that the Bi nanotubes are highly oriented and single crystalline. Electronic transport measurements proved that there is a metal–semiconductor transition of Bi nanotube arrays with the decrease of the wall thickness of the nanotubes, and this transition depends only on the wall thickness and is independent of the diameter of the nanotubes. The quantum confinement effect is believed to play an important role in determining transport properties. The Bi nanotubes may find applications in thermoelectric nanodevices.

Diameter-depended thermal expansion properties of Bi nanowire arrays

The lattice parameter of bismuth nanowires has been measured using the in situ high-temperature x-ray diffraction method. Single-crystalline Bi nanowire arrays with the diameters from 10nmto250nm have been fabricated within the porous anodic alumina membranes by a pulsed electrodeposition technique. Different temperature dependencies of lattice parameter and thermal expansion coefficient were found for Bi nanowires with different diameters, and there is a transition from positive thermal expansion coefficient at low temperature to negative one at high temperature, and the transition temperature shifts to high temperature with the increase in the diameter of Bi nanowires.

Base editing with a Cpf1–cytidine deaminase fusion

The targeting range of CRISPR–Cas9 base editors (BEs) is limited by their G/C-rich protospacer-adjacent motif (PAM) sequences. To overcome this limitation, we developed a CRISPR–Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and catalyzes C-to-T conversion in human cells, while inducing low levels of indels, non-C-to-T substitutions and off-target editing.

Highly efficient and precise base editing in discarded human tripronuclear embryos

CRISPR/Cas9 is a powerful tool for genome editing (Komor et al., 2017). Recently, it has been employed in several attempts to edit the human embryos (Liang et al., 2015; Kang et al., 2016; Tang et al., 2017). A major technical concern particularly relevant in studies involving human embryos is the potential off-target effects (Callaway, 2016; Plaza Reyes and Lanner, 2017). Consequently, development of safer genome editing strategy in human embryos is highly anticipated (Cyranoski and Reardon, 2015). The offtarget mutation result in part from Cas9-mediated double strand break (DSB) of DNA. Recently, base editing (BE) without the introduction of DSB has been achieved. The key design for BE is to use a catalytically inactive Cas9 to recruit the cytidine deaminase APOBEC to target sequences, leading to conversion of C to T within a window of approximately five nucleotides (Komor et al., 2016). Therefore, BE is apparently determined by additional features of the target sequence and offers a potentially safer approach for genome editing. Here we report the initial technical assessment of applying BE3, base editor 3 (Komor et al., 2016), in discarded human tripronuclear embryos. We targeted two human gene sites, HEK293 site 4 and RNF2 (Komor et al., 2016). BE3 and sgRNAs were prepared in vitro as described (Shen et al., 2014), and microinjected into the cytoplasm of the tripronuclear zygotes with the concentration of one hundred nanogram BE3 and fifty nanogram sgRNA per microliter. The zygotes were collected 48 h after microinjection, with the embryos containing different numbers of cells ranging from 1 to 8 (Table S1). In total, 8 zygotes for each of the two targets (#1–8 for HEK293 site 4, #9–16 for RNF2) were collected (Fig. 1A). Whole genome of each individual sample was amplified and used as the template for further analysis. To detect the efficiency of base editing, the region around the target sites was amplified and analyzed initially by the T7EN1 cleavage assay. For HEK293 site 4, we did not detect any cleavage bands in any of the samples (Fig. S1A). However, sequencing of the bulk PCR products revealed C to Tconversion at the sixteenth base distal from the PAM in 7 of the samples (#1–6, #8) (Figs. 1B and S1B), which is in accordance with the original report in human cell lines (Komor et al., 2016). We cloned 3 (#1–3) of the 8 bulk PCR products and sequenced multiple colonies from each primary product. For PCR products #2 and #3, each clone sequenced displayed C to T substitution, while PCR product #1 yielded one wildtype genotype besides the identical mutation genotypes (Fig. S1C), indicating highly efficient editing. To more carefully analyze the on-target editing effects, deep sequencing was applied to samples #2 and #3. In total, more than 3 M clean reads for each sample were generated. The results showed that only the 16th nucleotide distal from the PAM completely carried C to T conversion with the efficiency as high as 0.97 for sample #3, and 0.99 for sample #2. No other nucleotide alteration was detected (Fig. 1C). Besides, no on-target indel was found (Table S2). These results demonstrated the BE led to highly precise and efficient genome editing in human embryos. The same tests were performed for RNF2. T7EN1 cleavage bands were detected in 7 out of the 8 samples (#9–13, #15–16) (Fig. S2A). Sanger sequencing of PCR products confirmed C to T conversion in the 7 samples with cleavage (Figs. 1D and S2B). To further analyze the editing, 3 samples (#10–12) were selected for genotyping by TA cloning and subsequent sequencing. As reported before (Komor et al., 2016), in most cases, 2 cytosines (at the 18th and 15th nucleotide distal from the PAM) were simultaneously mutated to T, and triple C to T conversion (at the 18th, 15th, and 9th nucleotide distal from the PAM) also occurred (sample #10 and #12) (Fig. S2C). Collectively, these results demonstrated highly efficient and precise on-target base editing by BE3 in human embryos. We next tried to mutate the two genes simultaneously in the tripronuclear zygotes. To avoid possible toxicity, the concentration of each sgRNA was lowered to 25 nanogram per microliter. Nine embryos (#17–25) were collected and the target sites were analyzed by sequencing (Fig. 1A). For HEK293 site 4, the expected substitution in the sixteenth base distal from the PAM was observed in all samples (Fig. S3A), although the wild type genotype was also detectable in a few samples (Fig. S3B). A sample (#18) was randomly selected for on-target analysis by deep sequencing. The results showed that the conversion rate in the 16th C was about 0.68, which was consistent with the results of

How does spallation microdamage nucleate in bulk amorphous alloys under shock loading

Specially designed plate-impact experiments have been conducted on a Zr-based amorphous alloy using a single-stage light gas gun. To understand the microdamage nucleation process in the material, the samples are subjected to dynamic tensile loadings of identical amplitude (similar to 3.18 GPa) but with different durations (83-201 ns). A cellular pattern with an equiaxed shape is observed on the spallation surface, which shows that spallation in the tested amorphous alloy is a typical ductile fracture and that microvoids have been nucleated during the process. Based on the observed fracture morphologies of the spallation surface and free-volume theory, we propose a microvoid nucleation model of bulk amorphous alloys. It is found that nucleation of microvoids at the early stage of spallation in amorphous alloys results from diffusion and coalescence of free volume, and that high mean tensile stress plays a dominant role in microvoid nucleation

Ductile-to-brittle transition in spallation of metallic glasses

In this paper, the spallation behavior of a binary metallic glass Cu50Zr50 is investigated with molecular dynamics simulations. With increasing the impact velocity, micro-voids induced by tensile pulses become smaller and more concentrated. The phenomenon suggests a ductile-to-brittle transition during the spallation process. Further investigation indicates that the transition is controlled by the interaction between void nucleation and growth, which can be regarded as a competition between tension transformation zones (TTZs) and shear transformation zones (STZs) at atomic scale. As impact velocities become higher, the stress amplitude and temperature rise in the spall region increase and micro-structures of the material become more unstable. Therefore, TTZs are prone to activation in metallic glasses, leading to a brittle behavior during the spallation process

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.

A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.CRISPR-based base editors allow for single nucleotide genome editing in a range of organisms. Here the authors demonstrate the in vivo generation of mouse models carrying clinically relevant mutations using C→T and A→G editors.

Generation of fertile offspring from Kit(w)/Kit(wv) mice through differentiation of gene corrected nuclear transfer embryonic stem cells.

Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kitw/Kitwv mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kitw/Kitwv somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kitw/Kitwv infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.

APOBEC3 induces mutations during repair of CRISPR–Cas9-generated DNA breaks

The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR–Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR–Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.The APOBEC-AID family of cytidine deaminases target single-stranded nucleic acids for cytidine-to-uridine deamination and can thereby affect DNA repair processes that occur during CRISPR–Cas9-mediated genome editing.

Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos

There are urgent demands for efficient treatment of heritable genetic diseases. The base editing technology has displayed its efficiency and precision in base substitution in human embryos, providing a potential early-stage treatment for genetic diseases. Taking advantage of this technology, we corrected a Marfan syndrome pathogenic mutation, FBN1T7498C. We first tested the feasibility in mutant cells, then successfully achieved genetic correction in heterozygous human embryos. The results showed that the BE3 mediated perfect correction at the efficiency of about 89%. Importantly, no off-target and indels were detected in any tested sites in samples by high-throughput deep sequencing combined with whole-genome sequencing analysis. Our study therefore suggests the efficiency and genetic safety of correcting a Marfan syndrome (MFS) pathogenic mutation in embryos by base editing.