BackgroundMany applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.ResultsIn the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.ConclusionsNHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1518-x) contains supplementary material, which is available to authorized users.

Phosphorylated histone H2AX, termed ‘γH2AX’, mediates the chromatin response to DNA double strand breaks (DSBs) in mammalian cells. H2AX deficiency increases the numbers of unrepaired DSBs and translocations, which are partly associated with defects in non-homologous end joining (NHEJ) and contributing to genomic instability in cancer. However, the role of γH2AX in NHEJ of general DSBs has yet to be clearly defined. Here, we showed that despite little effect on overall NHEJ efficiency, H2AX deficiency causes a surprising bias towards accurate NHEJ and shorter deletions in NHEJ products. By analyzing CRISPR/Cas9-induced NHEJ and by using a new reporter for mutagenic NHEJ, we found that γH2AX, along with its interacting protein MDC1, is required for efficient classical NHEJ (C-NHEJ) but with short deletions and insertions. Epistasis analysis revealed that ataxia telangiectasia mutated (ATM) and the chromatin remodeling complex Tip60/TRRAP/P400 are essential for this H2AX function. Taken together, these data suggest that a subset of DSBs may require γH2AX-mediated short-range nucleosome repositioning around the breaks to facilitate C-NHEJ with loss of a few extra nucleotides at NHEJ junctions. This may prevent outcomes such as non-repair and translocations, which are generally more destabilizing to genomes than short deletions and insertions from local NHEJ.

The potential of adult human adipose tissue stem cells (hASCs) to differentiate into hepatocytes has generated much excitement over the possible use of hASCs in therapeutic applications. An understanding of the molecular mechanisms that underlie the plasticity of hASCs toward hepatocytes will help to make this possibility a reality. Herein, we show that a homogenous population of hASCs characterized by a high level of CD73, CD90, and CD105 express the pluripotent transcription factors OCT4, SOX2, NANOG, and SALL4 under proliferation conditions. A high level of activin A allows for hASCs acquiring the fate of definitive endoderm (DE) cells and expressing the specific transcription factors HEX, FOXA2, SOX17, and GATA4 synchronously. Using a reproducible three-stage method by mimicking liver embryogenesis, hASCs were directed to differentiate into functional hepatocytes. In the first stage, hASCs were induced to become DE cells by 2 days cultured in serum-free medium and 3 days of activin A treatment. Next, the presence of fibroblast growth factor (FGF) 4 and bone morphogenetic protein (BMP) 2 in the medium for 5 days induced efficient hepatic differentiation from DE cells. After 10 days of further maturated by the sequential exposure to hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone (DEX), the hASC-derived hepatocytes expressed mature hepatocytes marker and exhibited functional characterization, including albumin secretion, glycogen storage, urea production, activity of drug transporters, and cytochrome P450 activity. These findings will be useful for the implementation of hASC-derived hepatocytes in therapeutic purposes, metabolic analyses, drug toxicity screening, and studies of hepatocyte function.

Background Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. Results In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. Conclusions Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Currently, the key challenge in triboelectric nanogenerators (TENGs) is how to efficiently enhance the surface charge density. Here, a new strategy is proposed to increase the surface charge density by comprehensively utilizing solar energy and tidal energy, and a bioinspired photoelectric-electromechanical integrated TENG (Pem-iTENG) is developed. This enhancement of output performance is greatly attributed to the accumulation of photoelectrons from photocatalysis and the triboelectric negative charges from contact electrification. Pem-iTENG shows a maximal open-circuit voltage of 124.2 V and a maximal short-circuit current density of 221.6 μA cm−2 under tidal wave and sunlight, an improvement by nearly a factor of 10 over that of reported TENGs based on solid-liquid contact electrification. More importantly, it exhibits a high energy conversion efficiency according to the evaluation method for solar cells. This work provides insights into development of high-performance TENGs by using different natural energy sources.

Weight loss and cachexia are common problems in colorectal cancer patients; thus, parenteral and enteral nutrition support play important roles in cancer care. However, the impact of nonessential amino acid components of nutritional intake on cancer progression has not been fully studied. In this study, we discovered that gastrointestinal cancer patients who received cysteine as part of the parenteral nutrition had shorter overall survival (P < 0.001) than those who did not. Cystine indeed robustly promotes colon cancer cell growth in vitro and in immunodeficient mice, predominately by inhibiting SESN2 transcription via the GCN2-ATF4 axis, resulting in mTORC1 activation. mTORC1 inhibitors Rapamycin and Everolimus block cystine-induced cancer cell proliferation. In addition, cystine confers resistance to oxaliplatin and irinotecan chemotherapy by quenching chemotherapy-induced reactive oxygen species via synthesizing glutathione. We demonstrated that dietary deprivation of cystine suppressed colon cancer xenograft growth without weight loss in mice and boosted the antitumor effect of oxaliplatin. These findings indicate that cyst(e)ine, as part of supplemental nutrition, plays an important role in colorectal cancer and manipulation of cyst(e)ine content in nutritional formulations may optimize colorectal cancer patient survival.

In this paper, a kind of green triboelectric nano-generator based on natural degradable cellulose is proposed. Different kinds of regenerated cellulose composite layers are prepared by a blending doping method, and then assembled with poly(tetrafluoroethylene) (PTFE) thin films to form tribioelectric nanogenerator (TENG). The results show that the open circuit output voltage and the short circuit output current using a pure cellulose membrane is 7.925 V and 1.095 μA. After adding a certain amount of polyamide (PA6)/polyvinylidene fluoride (PVDF)/barium titanate (BaTiO3), the open circuit output voltage peak and the peak short circuit output current increases by 254.43% (to 20.155 V) and 548.04% (to 6.001 μA). The surface morphology, elemental composition and functional group of different cellulose layers are characterized by Scanning Electronic Microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and tested by the electrochemical analyze. Moreover, after multiple assembly and rectification processing, the electrical output performance shows that the peak value of open-circuit output voltage and the peak value of short circuit output current increases by 132.06% and 116.13%. Within 500 s of the charge-discharge test, the single peak charge reached 3.114 V, and the two peak charges reached 3.840 V. The results demonstrate that the nano-generator based on cellulose showed good stability and reliability, and the application and development of natural biomaterials represented by cellulose are greatly promoted in miniature electronic sensing area.

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