CRISPR-clear imaging of melanin-rich B16-derived solid tumors

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature

Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides

Abstract We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5’ nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity

CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Forster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications. c.2018 The Author

CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Forster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.

Additional file 1: Figure S1. of Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides

Comparison of editing efficiencies of Cas9-WT and high-fidelity Cas9 variants using gX19 and gX20 sgRNAs in HEK293T cells. Figure S2. Comparison of relative indel frequencies of Cas9 variants normalized to that of Cas9-WT in HeLa cells. Figure S3. Comparison of indel frequencies of HH-X20, HH-gX19, and gX19 sgRNAs in combination with Cas9-WT and Cas9 variants in HEK293T cells. Figure S4. Specificities of high-fidelity Cas9 variants in combination with HH-X20 sgRNA in HEK293T cells. Table S1. Indel frequencies of Cas9-WT, eCas9-1.1, and Cas9-HF1 combined with gX19 sgRNAs at 26 target sites with an HX19 sequence. Table S2. Comparison of Cas9-WT and Cas9 variants using gX19 sgRNAs at 26 target sites with an HX19 sequence. Table S3. Comparison of indel frequencies of HH-X20, HH-gX19, and gX19 sgRNAs in combination with Cas9-WT and Cas9 variants in HeLa cells. Table S4. Comparison of indel frequencies of HH-X20, HH-gX19, and gX19 sgRNAs in combination with Cas9-WT and Cas9 variants in HEK293T cells. (PDF 497 kb

CRISPR-clear imaging of melanin-rich B16-derived solid tumors

Tyrosinase CRISPR knock out in the B16 melanoma cell line eliminates melanin production, with the lack of pigment not affecting tumour engraftment or growth and allowing for 3D imaging after clearing by PACT

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering approximate to 2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim11sciescopu

Anode-Less All-Solid-State Batteries Operating at Room Temperature and Low Pressure

Anode-less all-solid-state batteries (ASSBs) are being targeted for next-generation electric mobility owing to their superior energy density and safety as well as the affordability of their materials. However, because of the anode-less configuration, it is nontrivial to simultaneously operate the cell at room temperature and low pressure as a result of the sluggish reaction kinetics of lithium (de)plating and the formation of interfacial voids. This study overcomes these intrinsic challenges of anode-less ASSBs by introducing a dual thin film consisting of a magnesium upper layer with a Ti3C2Tx MXene buffer layer underneath. The Mg layer enables reversible Li plating and stripping at room temperature by reacting with Li via a (de)alloying reaction with a low reaction barrier. The MXene buffer layer maintains the electrolyte-electrode interface by inhibiting the formation of voids even at low pressure of 2 MPa owing to the high ductility of MXene. This study highlights the importance of a combined chemical and mechanical approach when designing anode-less electrodes for practical adaptation for anode-less ASSBs

Myofibroblast in the ligamentum flavum hypertrophic activity

Majority of the previous studies compared lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) patients for analyses of LFH. However, the separation of normal/hypertrophied LF has often been ambiguous and the severity of hypertrophic activity differed. Here, we present a novel analysis scheme for LFH in which myofibroblast is proposed as a major etiological factor for LFH study. Seventy-one LF patient tissue samples were used for this study. Initially, mRNA levels of the samples were assessed by qRT-PCR: angiopoietin-like protein-2 (ANGPTL2), transforming growth factor-beta1 (TGF-beta 1), vascular endothelial growth factor (VEGF), interleukin-6, collagen-1, 3, 4, 5, and 11, and elastin. Myofibroblasts were detected by immune stain using alpha-smooth muscle actin (alpha SMA) as a marker. To study the myofibroblast in TGF-beta pathway, LF tissues were analyzed for protein levels of alpha SMA/TGF-beta 1 by Western blot. In addition, from LF cells cultured with exogenous TGF-beta 1 conditioned medium, expression of alpha SMA/collagen-1 was assessed and the cell morphology was identified. The comparative analysis of mRNA expression levels (LSS vs LDH) failed to show significant differences in TGF-beta 1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-beta 1, and collagen-1 and 3, which represent common trends in hypertrophic activity (p < 0.05). We detected myofibroblast in the patient samples by alpha SMA staining, and the protein levels of alpha SMA were positively correlated with TGF-beta 1. In LF cell culture, exogenous TGF-beta 1 upregulated alpha SMA/collagen-1 mRNA levels and facilitated trans-differentiation to myofibroblast. We conclude that the transition of fibroblast to myofibroblasts via TGF-beta pathway is a key linker between inflammation and fibrosis in LFH mechanism. (c) Springer-Verlag Berlin Heidelberg 20171231sciescopu