CWAS-Plus: estimating category-wide association of rare noncoding variation from whole-genome sequencing data with cell-type-specific functional data.

Variants in cis-regulatory elements link the noncoding genome to human pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS), enhances noncoding variant analysis by integrating both whole-genome sequencing (WGS) and user-provided functional data. With simplified parameter settings and an efficient multiple testing correction method, CWAS-Plus conducts the CWAS workflow 50 times faster than CWAS, making it more accessible and user-friendly for researchers. Here, we used a single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type-specific enhancers and promoters. Examining autism spectrum disorder WGS data (n = 7280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimer's disease WGS data (n = 1087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Plus's utility in genomic disorders and scalability for processing large-scale WGS data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/, respectively.

SpatialSPM: statistical parametric mapping for the comparison of gene expression pattern images in multiple spatial transcriptomic datasets.

Spatial transcriptomic (ST) techniques help us understand the gene expression levels in specific parts of tissues and organs, providing insights into their biological functions. Even though ST dataset provides information on the gene expression and its location for each sample, it is challenging to compare spatial gene expression patterns across tissue samples with different shapes and coordinates. Here, we propose a method, SpatialSPM, that reconstructs ST data into multi-dimensional image matrices to ensure comparability across different samples through spatial registration process. We demonstrated the applicability of this method by kidney and mouse olfactory bulb datasets as well as mouse brain ST datasets to investigate and directly compare gene expression in a specific anatomical region of interest, pixel by pixel, across various biological statuses. Beyond traditional analyses, SpatialSPM is capable of generating statistical parametric maps, including T-scores and Pearson correlation coefficients. This feature enables the identification of specific regions exhibiting differentially expressed genes across tissue samples, enhancing the depth and specificity of ST studies. Our approach provides an efficient way to analyze ST datasets and may offer detailed insights into various biological conditions.

Bright Bifacial White-Light Illumination by Highly Deformable Electroluminescent Devices Based on Transparent Ionic-Hydrogel Electrodes and Quantum-Dot Color Conversion.

Deformable alternating-current electroluminescent (ACEL) devices are of increasing interest because of their potential to drive innovation in soft optoelectronics. Despite the research focus on efficient white ACEL devices, achieving deformable devices with high luminance remains difficult. In this study, this challenge is addressed by fabricating white ACEL devices using color-conversion materials, transparent and durable hydrogel electrodes, and high-k nanoparticles. The incorporation of quantum dots enables the highly efficient generation of red and green light through the color conversion of blue electroluminescence. Although the ionic-hydrogel electrode provides high toughness, excellent light transmittance, and superior conductivity, the luminance of the device is remarkably enhanced by the incorporation of a high-k dielectric, BaTiO3. The fabricated ACEL device uniformly emits very bright white light (489 cd m-2) with a high color-rendering index (91) from both the top and bottom. The soft and tough characteristics of the device allow seamless operation in various deformed states, including bending, twisting, and stretching up to 400%, providing a promising platform for applications in a wide array of soft optoelectronics.

READRetro: natural product biosynthesis predicting with retrieval-augmented dual-view retrosynthesis.

Plants, as a sessile organism, produce various secondary metabolites to interact with the environment. These chemicals have fascinated the plant science community because of their ecological significance and notable biological activity. However, predicting the complete biosynthetic pathways from target molecules to metabolic building blocks remains a challenge. Here, we propose retrieval-augmented dual-view retrosynthesis (READRetro) as a practical bio-retrosynthesis tool to predict the biosynthetic pathways of plant natural products. Conventional bio-retrosynthesis models have been limited in their ability to predict biosynthetic pathways for natural products. READRetro was optimized for the prediction of complex metabolic pathways by incorporating cutting-edge deep learning architectures, an ensemble approach, and two retrievers. Evaluation of single- and multi-step retrosynthesis showed that each component of READRetro significantly improved its ability to predict biosynthetic pathways. READRetro was also able to propose the known pathways of secondary metabolites such as monoterpene indole alkaloids and the unknown pathway of menisdaurilide, demonstrating its applicability to real-world bio-retrosynthesis of plant natural products. For researchers interested in the biosynthesis and production of secondary metabolites, a user-friendly website (https://readretro.net) and the open-source code of READRetro have been made available.

3D printable and mechanically tunable hydrogels achieved through hydrophobic and ionic interactions.

Thermal stiffening materials are commonly applied in the aerospace and automotive industries, among others, since their dimensional stabilities and stiffness characteristics improve at high temperatures. In this study, temperature-triggered modulus-tunable hydrogels were prepared by combining Pluronic F-127 with charged polymers. Pluronic F-127, a triblock copolymer micelle, provided three-dimensional printing capabilities of fine resolution with high viscosity, while hydrophobic and ionic interactions among polymer networks provided thermal stiffening. The hydrogel ink's printability was demonstrated by successfully creating complex 3D structures. A calcium ion carrying a hydrophobic propionate and carboxylate group in polymer chains was used to form additional physical crosslinking at high temperature, ultimately leading to the thermal stiffening effect without volume change. The thermal stiffening behavior was found to be fully reversible and repeatable. Finally, to demonstrate the versatility of this work, graphene oxide was added to produce a light-controllable modulus based on its photothermal properties.

Development of finely tuned liposome nanoplatform for macrophage depletion.

BACKGROUND: Immunotherapy with clodronate-encapsulated liposomes, which induce macrophage depletion, has been studied extensively. However, previously reported liposomal formulation-based drugs (Clodrosome® and m-Clodrosome®) are limited by their inconsistent size and therapeutic efficacy. Thus, we aimed to achieve consistent therapeutic effects by effectively depleting macrophages with uniform-sized liposomes. RESULTS: We developed four types of click chemistry-based liposome nanoplatforms that were uniformly sized and encapsulated with clodronate, for effective macrophage depletion, followed by conjugation with Man-N3 and radiolabeling. Functionalization with Man-N3 improves the specific targeting of M2 macrophages, and radioisotope labeling enables in vivo imaging of the liposome nanoplatforms. The functionalized liposome nanoplatforms are stable under physiological conditions. The difference in the biodistribution of the four liposome nanoplatforms in vivo were recorded using positron emission tomography imaging. Among the four platforms, the clodronate-encapsulated mannosylated liposome effectively depleted M2 macrophages in the normal liver and tumor microenvironment ex vivo compared to that by Clodrosome® and m-Clodrosome®. CONCLUSION: The newly-developed liposome nanoplatform, with finely tuned size control, high in vivo stability, and excellent ex vivo M2 macrophage targeting and depletion effects, is a promising macrophage-depleting agent.

IG-MYC + neoplasms with precursor B-cell phenotype are molecularly distinct from Burkitt lymphomas.

The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein "preBLL"). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq.

We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

Cpf1-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cpf1.

Summary: Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale. One can easily access the data using a straightforward web interface, and using the powerful collections feature one can easily design gRNAs for thousands of genes in short time. Availability and implementation: Free access at http://www.rgenome.net/cpf1-database/. Contact: sangsubae@hanyang.ac.kr.

Web-based design and analysis tools for CRISPR base editing.

BACKGROUND: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed. RESULTS: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (< 1 GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively. CONCLUSION: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Cas-analyzer: an online tool for assessing genome editing results using NGS data.

Genome editing with programmable nucleases has been widely adopted in research and medicine. Next generation sequencing (NGS) platforms are now widely used for measuring the frequencies of mutations induced by CRISPR-Cas9 and other programmable nucleases. Here, we present an online tool, Cas-Analyzer, a JavaScript-based implementation for NGS data analysis. Because Cas-Analyzer is completely used at a client-side web browser on-the-fly, there is no need to upload very large NGS datasets to a server, a time-consuming step in genome editing analysis. Currently, Cas-Analyzer supports various programmable nucleases, including single nucleases and paired nucleases. AVAILABILITY AND IMPLEMENTATION: Free access at http://www.rgenome.net/cas-analyzer/ CONTACT: sangsubae@hanyang.ac.kr or jskim01@snu.ac.krSupplementary information: Supplementary data are available at Bioinformatics online.

Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations.

Cancer drug screening in patient-derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix-dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy-based assay to resolve drug-induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug-induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.

Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9.

MOTIVATION: CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult. RESULTS: We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users. AVAILABILITY AND IMPLEMENTATION: Free access at http://www.rgenome.net/cas-database/ CONTACT: sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer.

Colon cancer is one of the most common types of cancer, and it has recently become a leading cause of death worldwide. Among colon cancers, the v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated form is notorious for its non-druggable features. Cetuximab, a monoclonal antibody that binds to the epidermal growth factor receptor, has been introduced as an antitumor therapy; however, secondary resistance and side effects significantly limit its effective use in these cancers. In this study, we prepared Phellinuslinteus on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. The combined treatment of PBR extract and cetuximab suppressed SW480 cell viability/proliferation, with the cells exhibiting altered cellular morphology and clonogenic potential. AnnexinV-fluorescein isothiocyanate/propidium iodide-stained flow cytometry and Western blotting were performed, and PBR extract combined with cetuximab treatment increased apoptosis of the SW480 cells and suppressed their KRAS protein expression. The potential of PBR as a synergistic anticancer agent was further investigated in a tumor-xenografted mouse model. Tumor growth was significantly suppressed with PBR extract and cetuximab co-treatment. In conclusion, PBR increased the sensitivity of KRAS-mutated colon cancer cells to cetuximab, which indicates the potential use of PBR as a medical food against colon cancer.

Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites.

UNLABELLED: We present Cas-Designer, a user-friendly program to aid researchers in choosing appropriate target sites in a gene of interest for type II CRISPR/Cas-derived RNA-guided endonucleases, which are now widely used for biomedical research and biotechnology. Cas-Designer rapidly provides the list of all possible guide RNA sequences in a given input DNA sequence and their potential off-target sites including bulge-type sites in a genome of choice. In addition, the program assigns an out-of-frame score to each target site to help users choose appropriate sites for gene knockout. Cas-Designer shows the results in an interactive table and provides user-friendly filter functions. AVAILABILITY AND IMPLEMENTATION: Free access at http://rgenome.net/cas-designer/.

Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases.

SUMMARY: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website. AVAILABILITY AND IMPLEMENTATION: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder. CONTACT: baesau@snu.ac.kr or jskim01@snu.ac.kr.