scSNV-seq: high-throughput phenotyping of single nucleotide variants by coupled single-cell genotyping and transcriptomics.

CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations including single nucleotide variants (SNVs) associated with diseases. Interpretation of these data is currently limited as genotypes cannot be accurately inferred from guide RNA identity alone. scSNV-seq overcomes this limitation by coupling single-cell genotyping and transcriptomics of the same cells enabling accurate and high-throughput screening of SNVs. Analysis of variants across the JAK1 gene with scSNV-seq demonstrates the importance of determining the precise genetic perturbation and accurately classifies clinically observed missense variants into three functional categories: benign, loss of function, and separation of function.

Screening for functional regulatory variants in open chromatin using GenIE-ATAC.

Understanding the effects of genetic variation in gene regulatory elements is crucial to interpreting genome function. This is particularly pertinent for the hundreds of thousands of disease-associated variants identified by GWAS, which frequently sit within gene regulatory elements but whose functional effects are often unknown. Current methods are limited in their scalability and ability to assay regulatory variants in their endogenous context, independently of other tightly linked variants. Here, we present a new medium-throughput screening system: genome engineering based interrogation of enhancers assay for transposase accessible chromatin (GenIE-ATAC), that measures the effect of individual variants on chromatin accessibility in their endogenous genomic and chromatin context. We employ this assay to screen for the effects of regulatory variants in human induced pluripotent stem cells, validating a subset of causal variants, and extend our software package (rgenie) to analyse these new data. We demonstrate that this methodology can be used to understand the impact of defined deletions and point mutations within transcription factor binding sites. We thus establish GenIE-ATAC as a method to screen for the effect of gene regulatory element variation, allowing identification and prioritisation of causal variants from GWAS for functional follow-up and understanding the mechanisms of regulatory element function.

Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.

CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Posttranscriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22 nt) noncoding RNAs capable of specifically targeting the miRNA-induced silencing complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct in situ interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering (Bassett G et al., Nat Commun 5, 4640, 2014). In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).

Effect of Traditional Chinese Medicine on Psoriasis Vulgaris: A Meta-Analysis and Systematic Review.

BACKGROUND: Psoriasis is a chronic, inflammatory autoimmune skin disease that affects 2-3% of the world's population. Lesions are mainly found on the limbs, trunk, and scalp, but may also affect other parts of the body, and the cause is not yet known. The chronic and relapsing nature of psoriasis makes it one of the most complex and important diseases in current dermatology research. METHODS: The search was conducted using PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Internet, Wanfang Data, VIP journals database, and Chinese biomedical literature database (CBM). The retrieval time limit was from the establishment of the database to January 2021. The quality of the selected literature was evaluated, and ReView Manager 5.3 was used for meta-analysis after randomized controlled trials were filtered. RESULTS: Finally, 16 randomized controlled trials involving 1,967 patients were included. The total effective rate (OR = 3.68, 95% CI [2.73, 4.95], p < 0.00001), cure rate (OR = 2.01, 95% CI [1.62, 2.49], p < 0.00001), and PASI score (OR = -1.83, 95% CI [-2.39, -1.26], p < 0.00001) of the traditional Chinese medicine (TCM) were superior to the Diyin tablet. CONCLUSION: In the treatment of psoriasis, TCM shows higher efficacy than the Diyin tablet. However, due to the limitations of the included literature, we still need more double-blind, placebo-controlled trials with large samples and multiple centers to provide high-quality clinical evidence.

Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells.

We explored human induced pluripotent stem cells (hiPSCs) derived from different tissues to gain insights into genomic integrity at single-nucleotide resolution. We used genome sequencing data from two large hiPSC repositories involving 696 hiPSCs and daughter subclones. We find ultraviolet light (UV)-related damage in ~72% of skin fibroblast-derived hiPSCs (F-hiPSCs), occasionally resulting in substantial mutagenesis (up to 15 mutations per megabase). We demonstrate remarkable genomic heterogeneity between independent F-hiPSC clones derived during the same round of reprogramming due to oligoclonal fibroblast populations. In contrast, blood-derived hiPSCs (B-hiPSCs) had fewer mutations and no UV damage but a high prevalence of acquired BCOR mutations (26.9% of lines). We reveal strong selection pressure for BCOR mutations in F-hiPSCs and B-hiPSCs and provide evidence that they arise in vitro. Directed differentiation of hiPSCs and RNA sequencing showed that BCOR mutations have functional consequences. Our work strongly suggests that detailed nucleotide-resolution characterization is essential before using hiPSCs.

A blood-based miRNA signature with prognostic value for overall survival in advanced stage non-small cell lung cancer treated with immunotherapy.

Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune-related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ~30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction. We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well-characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37-4.19; P < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signaling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining.

Prediction of exacerbation frequency of AECOPD based on next-generation sequencing and its relationship with imbalance of lung and gut microbiota: a protocol of a prospective cohort study.

INTRODUCTION: Patients with frequent acute exacerbation phenotype chronic obstructive pulmonary disease (AECOPD) have a higher hospitalisation rate than infrequent exacerbation, the disease progresses quickly and treatment is more difficult. At present, it is impossible to predict patients with COPD with frequent acute exacerbation phenotypes. The composition of the lower respiratory tract flora and the intestinal flora is closely related to AECOPD, but the specific association mechanism between them is not very clear. This study used metagenomic next-generation sequencing (mNGS) technology to explore the microbial characteristics of the intestinal tract and airways of patients with COPD, and analyse the correlation between the sequencing results and inflammatory factors, immune factors and nutritional factors. METHODS AND ANALYSIS: This will be a prospective cohort study. We intend to recruit 152 patients with stable COPD. In the baseline, we will detect the participants' induced sputum and faecal flora through mNGS, and changes in blood immune levels, and the patient's condition is evaluated. Every 2 months, we will check the number of acute exacerbation through the phone range. After 12 months, we will check again the changes in the blood immune level, evaluate the patient's condition and count the number of episodes. ETHICS AND DISSEMINATION: This study has been approved by the ethics committee of Guangdong Provincial Hospital of Traditional Chinese Medicine (approval number ZF2019-219-03). The results of the study will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (ChiCTR2000032870).

INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing.

We present INSIGHT [isothermal NASBA (nucleic acid sequence-based amplification) sequencing-based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing-based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Post-transcriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22nt) noncoding RNAs capable of specifically targeting the miRNA-induced-silencing-complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering. In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks, and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).

Experimental bariatric surgery in rats generates a cytotoxic chemical environment in the gut contents.

Bariatric surgery, also known as metabolic surgery, is an effective treatment for morbid obesity, which also offers pronounced metabolic effects including the resolution of type 2 diabetes and a decrease in cardiovascular disease and long-term cancer risk. However, the mechanisms of surgical weight loss and the long-term consequences of bariatric surgery remain unclear. Bariatric surgery has been demonstrated to alter the composition of both the microbiome and the metabolic phenotype. We observed a marked shift toward Gammaproteobacteria, particularly Enterobacter hormaechei, following Roux-en-Y gastric bypass (RYGB) surgery in a rat model compared with sham-operated controls. Fecal water from RYGB surgery rats was highly cytotoxic to rodent cells (mouse lymphoma cell line). In contrast, fecal water from sham-operated animals showed no/very low cytotoxicity. This shift in the gross structure of the microbiome correlated with greatly increased cytotoxicity. Urinary phenylacetylglycine and indoxyl sulfate and fecal gamma-aminobutyric acid, putrescine, tyramine, and uracil were found to be inversely correlated with cell survival rate. This profound co-dependent response of mammalian and microbial metabolism to RYGB surgery and the impact on the cytotoxicity of the gut luminal environment suggests that RYGB exerts local and global metabolic effects which may have an influence on long-term cancer risk and cytotoxic load.