Liu J

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.dCas9-mediated activation has been verified and widely used in vitro. Here the authors generated a potent in vivo activation platform and applied it to control the transcription of multiple genetic elements in the mammalian brain.

CRISPR/Cas9 – Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah−/− liver failure mice, offering an efficient approach for precisely targeted gene therapies.

High power buried sampled grating distributed feedback quantum cascade lasers

A novel index-coupled distributed feedback quantum cascade laser emitting at λu2009∼u20094.8u2009μm is demonstrated by a sampled grating. The coupling coefficient can be almost controlled arbitrarily according to the duty cycle of sampled grating. The additional supermodes caused by the sampled grating can be strongly suppressed by choosing a small sampling period, so that the supermodes are shifted apart from the gain curve. Single-mode emission without any significant disadvantages compared with uniform grating is achieved. Especially, this powerful approach presented here can be applied to achieve the performance with high power and low threshold simultaneously.

High-Pressure and High-Temperature in situ X-Ray Diffraction Study of FeP2 up to 70 GPa

The high-pressure and high-temperature structural behavior of FeP2 is investigated by means of synchrotron x-ray powder diffraction combined with a laser heating technique up to 70GPa and at least 1800 K. No phase transition of FeP2 occurs up to 68 GPa at room temperature. While a new phase of FeP2 assigned to the CuAl2-type structure (I4/mcm, Z = 4) is observed at 70 GPa after laser-heating. This new phase presents a quenchable property on decompression to ambient conditions. Our results update previous experimental data and are consistent with theoretical studies.

Pericentral hepatocytes produce insulin-like growth factor-2 to promote liver regeneration during selected injuries in mice

Liver regeneration (LR) happens after various types of injuries. Unlike the well‐studied LR caused by partial hepatectomy (PHx), there is accumulating evidence suggesting that LR during other injuries may result from unknown mechanisms. In this study, we found that insulin‐like growth factor 2 (IGF‐2) was drastically induced following the liver injuries caused by tyrosinemia or long‐term treatments of CCl4. However, this was not observed during the early phase of acute liver injuries after PHx or single treatment of CCl4. Remarkably, most IGF‐2‐expressing hepatocytes were located at the histological area around the central vein of the liver lobule after the liver injuries caused either in fumarylacetoacetate hydrolase–deficient mice or in CCl4 chronically treated mice. Hepatocyte proliferation in vivo was significantly promoted by induced IGF‐2 overexpression, which could be inhibited by adeno‐associated virus–delivered IGF‐2 short hairpin RNAs or linsitinib, an inhibitor of IGF‐2 signaling. Proliferating hepatocytes in vivo responded to IGF‐2 through both insulin receptor and IGF‐1 receptor. IGF‐2 also significantly promoted DNA synthesis of primary hepatocytes in vitro. More interestingly, the significantly induced IGF‐2 was also found to colocalize with glutamine synthetase in the region enriched with proliferating hepatocytes for the liver samples from patients with liver fibrosis. Conclusion: IGF‐2 is produced by pericentral hepatocytes to promote hepatocyte proliferation and repair tissue damage in the setting of chronic liver injury, which is distinct from the signaling that occurs post‐PHx. (Hepatology 2017;66:2002–2015)

Index-coupled surface porous grating distributed feedback quantum cascade laser

We report design of special low loss, index-coupled surface porous grating distributed feedback profile by using holographic lithography combined with electro-chemical etching technology. Room temperature continuous-wave (cw) operation of single-mode quantum cascade lasers emitting at λu2009∼u20097.6u2009μm has been achieved. Due to attenuate interplay between the surface plasmon waves and the waveguide waves, the waveguide loss of the lasers is significantly reduced, when compared to conventional surface metal/semiconductor grating devices. This resulted in an improved overall laser performance, such as a reduction of the threshold current density and an increase in cw output power.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

Multi-wavelength surface emitting quantum cascade laser based on equivalent phase shift

A novel surface emitting distributed feedback quantum cascade laser emitting around λu2009∼u20094.6u2009μm is demonstrated by employing an equivalent phase shift (EPS) of quarter-wave (λ/4). The EPS is fabricated through extending one sampling period by 50% in the center of a sampled Bragg grating. Single-lobed far-field radiation pattern with a low divergence angle of about 0.6°u2009×u200916.8° is obtained. Selective single-mode lasing with a mean side mode suppression ratio above 20u2009dB and wavelength coverage range of 72u2009nm is achieved simultaneously on a single wafer only by changing the sampling period.

High-Duty-Cycle Operation of GaAs/AlGaAs Quantum Cascade Laser above Liquid Nitrogen Temperature

We present a detailed study of lambda similar to 9.75 mu m GaAs/AIGaAs quantum cascade lasers. For a coated 2-mm-long and 40-mu m-wide laser, an optical power of 85 mu W is observed 95% duty cycle at 80 K. At a moderate driving pulse (1 kHz and 1% duty cycle), the device presents a peak power more than 20 mW even at 120 K. At 80 K, the fitted result of threshold current densities shows evidence of potential cw operation.