Yuxuan Wu

Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9

The CRISPR-Cas9 system has been employed to generate mutant alleles in a range of different organisms. However, so far there have not been reports of use of this system for efficient correction of a genetic disease. Here we show that mice with a dominant mutation in Crygc gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant allele. Correction occurred via homology-directed repair (HDR) based on an exogenously supplied oligonucleotide or the endogenous WT allele, with only rare evidence of off-target modifications. The resulting mice were fertile and able to transmit the corrected allele to their progeny. Thus, our study provides proof of principle for use of the CRISPR-Cas9 system to correct genetic disease.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Histone deacetylation promotes mouse neural induction by restricting Nodal-dependent mesendoderm fate.

Cell fate determination requires the cooperation between extrinsic signals and intrinsic molecules including transcription factors as well as epigenetic regulators. Nevertheless, how neural fate commitment is regulated by epigenetic modifications remains largely unclear. Here we show that transient histone deacetylation at epiblast stage promotes neural differentiation of mouse embryonic stem cells (mESCs). Histone deacetylase 1 (HDAC1) deficiency in mESCs partially phenocopies the inhibition of histone deacetylation in vitro, and displays reduced incorporation into neural tissues in chimeric mouse embryos in vivo. Mechanistic studies show that Nodal, which is repressed by histone deacetylation, is a direct target of HDAC1. Furthermore, the inhibition of histone deacetylation in the anterior explant of mouse embryos at E7.0 leads to Nodal activation and neural development repression. Thus, our study reveals an intrinsic mechanism that epigenetic histone deacetylation ensures neural fate commitment by restricting Nodal signalling in murine anterior epiblast ex vivo and mESC in vitro.

Parthenogenetic haploid embryonic stem cells efficiently support mouse generation by oocyte injection

Parthenogenetic haploid embryonic stem cells efficiently support mouse generation by oocyte injection

Generation and application of mammalian haploid embryonic stem cells

Haploid cells contain one set of chromosomes and are amenable for genetic analyses. In mammals, haploidy exists only in gametes. An intriguing question is whether haploid cells can be derived from gametes. Recently, by application of haploid cell enrichment using fluorescence‐activated cell sorting, stable haploid embryonic stem cells (haESCs) have been successfully derived from oocyte‐derived parthenogenetic and sperm‐derived androgenetic embryos from several species. Whilst both parthenogenetic and androgenetic (AG)‐haESCs enable whole‐genome genetic screening at the cellular level, such as screening of drug resistance or disease‐related genes, AG‐haESCs, after intracytoplasmic injection into oocytes, can also be used to produce alive semi‐cloned mice. Nevertheless, one major drawback associated with wild‐type AG‐haESCs is the very low birth rate of healthy semi‐cloned mice. Of interest, after inhibiting the expression of two paternally imprinted genes (H19 and Gtl2) in AG‐haESCs by removal of their differentially DNA methylated regions, double‐knockout AG‐haESCs can efficiently and stably support the generation of healthy semi‐cloned pups. Importantly, double‐knockout AG‐haESCs are feasible for multiple genetic manipulations, followed by efficient generation of semi‐cloned mice carrying multiple genetic traits; thus they could be used to validate candidate loci that have been identified in genome‐wide association studies of multigenic diseases by generation of mouse models carrying multiple alterations. Of note, by combining a CRISPR‐Cas9 library and double‐knockout AG‐haESCs, semi‐cloned mice carrying different mutant genes can be efficiently generated in one step, enabling functional mutagenic screening in mice. HaESCs, therefore, provide a powerful tool for genetic analyses in mammals at both the cellular and organismal levels.

Experimental study of the free spectral range (FSR) in FPI with a small plate gap

In this paper we investigate the variation of free spectral range (FSR) for the Fabry-Perot interferometer (FPI) consisting of mirrors with phase shift dispersion. The reflection phase shift on a mirror has been calculated employing the Transfer-Matrix Method and the values of FSR have been calculated under the condition of normal incidence of light beam. Fabry-Perot (FP) cavities have been fabricated employing bulk micromachining technology, and silicon wafers coated with multiplayer dielectric films were used as mirrors. FSR of these FP cavities have been experimentally measured. The experimental data match the calculated results very well. The conclusion is that FSR shortening effect must be taken into account for the FPIs with a small plate gap, as the finesse and the tunable range of tunable FPI can be affected by the shortening effect greatly.

THE ACTIVE OXYGEN ON THE LI/LA2O3 CATALYST SURFACE AND ITS CATALYTIC BEHAVIOR IN THE OXIDATIVE COUPLING OF METHANE

The coupling selectivity was greatly enhanced by adding Li to La2O3, compared with the single La2O3. The O2- species was found on the Li/La2O3 but not on the single La2O3. In low-temperature desorption, ethane desorbed from the Li/La2O3 but was not detected with the single La2O3. It is considered that the addition of Li gave rise to some basic sites which are favorable for the coupling reaction.