Nature Biotechnology

Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of experimental and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clinical diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and analytical approaches through the development of new nanopores, base-calling methods and experimental protocols tailored to particular applications.

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Wearable materials with embedded synthetic biology sensors for biomolecule detection

Peter Q. Nguyen, Luis R. Soenksen, Nina M. Donghia, Nicolaas M. Angenent-Mari, Helena de Puig, Ally Huang, Rose Lee, Shimyn Slomovic, Tommaso Galbersanini, Geoffrey Lansberry, Hani M. Sallum, Evan M. Zhao, James B. Niemi & James J. Collins

doi : 10.1038/s41587-021-00950-3

Nature Biotechnology volume 39, pages1366–1374 (2021)

Integrating synthetic biology into wearables could expand opportunities for noninvasive monitoring of physiological status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightweight, flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chemicals and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aqueous exposure events and report the presence of specific molecular targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current laboratory methods such as quantitative PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temperature within 90?min, requiring no user intervention other than the press of a button.

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Spatial transcriptomics at subspot resolution with BayesSpace

Edward Zhao, Matthew R. Stone, Xing Ren, Jamie Guenthoer, Kimberly S. Smythe, Thomas Pulliam, Stephen R. Williams, Cedric R. Uytingco, Sarah E. B. Taylor, Paul Nghiem, Jason H. Bielas & Raphael Gottardo

doi : 10.1038/s41587-021-00935-2

Nature Biotechnology volume 39, pages1375–1384 (2021)

Recent spatial gene expression technologies enable comprehensive measurement of transcriptomic profiles while retaining spatial context. However, existing analysis methods do not address the limited resolution of the technology or use the spatial information efficiently. Here, we introduce BayesSpace, a fully Bayesian statistical method that uses the information from spatial neighborhoods for resolution enhancement of spatial transcriptomic data and for clustering analysis. We benchmark BayesSpace against current methods for spatial and non-spatial clustering and show that it improves identification of distinct intra-tissue transcriptional profiles from samples of the brain, melanoma, invasive ductal carcinoma and ovarian adenocarcinoma. Using immunohistochemistry and an in silico dataset constructed from scRNA-seq data, we show that BayesSpace resolves tissue structure that is not detectable at the original resolution and identifies transcriptional heterogeneity inaccessible to histological analysis. Our results illustrate BayesSpace’s utility in facilitating the discovery of biological insights from spatial transcriptomic datasets.

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Volumetric interferometric lattice light-sheet imaging

Bin Cao, Simao Coelho, Jieru Li, Guanshi Wang & Alexandros Pertsinidis

doi : 10.1038/s41587-021-01042-y

Nature Biotechnology volume 39, pages1385–1393 (2021)

Live cell imaging with high spatiotemporal resolution and high detection sensitivity facilitates the study of the dynamics of cellular structure and function. However, extracting high-resolution 4D (3D space plus time) information from live cells remains challenging, because current methods are slow, require high peak excitation intensities or suffer from high out-of-focus background. Here we present 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that requires low excitation light levels and provides high background suppression and substantially improved volumetric resolution by combining 4Pi interferometry with selective plane illumination. We demonstrate that 3D-iLLS has an axial resolution and single-particle localization precision of 100?nm (FWHM) and <10?nm (1?), respectively. We illustrate the performance of 3D-iLLS in a range of systems: single messenger RNA molecules, nanoscale assemblies of transcription regulators in the nucleus, the microtubule cytoskeleton and mitochondria organelles. The enhanced 4D resolution and increased signal-to-noise ratio of 3D-iLLS will facilitate the analysis of biological processes at the sub-cellular level.

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Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore

Ploy N. Pratanwanich, Fei Yao, Ying Chen, Casslynn W. Q. Koh, Yuk Kei Wan, Christopher Hendra, Polly Poon, Yeek Teck Goh, Phoebe M. L. Yap, Jing Yuan Chooi, Wee Joo Chng, Sarah B. Ng, Alexandre Thiery, W. S. Sho Goh & Jonathan Göke

doi : 10.1038/s41587-021-00949-w

Nature Biotechnology volume 39, pages1394–1402 (2021)

RNA modifications, such as N6-methyladenosine (m6A), modulate functions of cellular RNA species. However, quantifying differences in RNA modifications has been challenging. Here we develop a computational method, xPore, to identify differential RNA modifications from nanopore direct RNA sequencing (RNA-seq) data. We evaluate our method on transcriptome-wide m6A profiling data, demonstrating that xPore identifies positions of m6A sites at single-base resolution, estimates the fraction of modified RNA species in the cell and quantifies the differential modification rate across conditions. We apply xPore to direct RNA-seq data from six cell lines and multiple myeloma patient samples without a matched control sample and find that many m6A sites are preserved across cell types, whereas a subset exhibit significant differences in their modification rates. Our results show that RNA modifications can be identified from direct RNA-seq data with high accuracy, enabling analysis of differential modifications and expression from a single high-throughput experiment.

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Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Ping Xu, Zhiheng Liu, Ying Liu, Huazheng Ma, Yiyuan Xu, Ying Bao, Shiyou Zhu, Zhongzheng Cao, Zeguang Wu, Zhuo Zhou & Wensheng Wei

doi : 10.1038/s41587-021-00944-1

Nature Biotechnology volume 39, pages1403–1413 (2021)

Canonical CRISPR–knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR–KO screens in various settings.

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Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning

Luke W. Koblan, Mandana Arbab, Max W. Shen, Jeffrey A. Hussmann, Andrew V. Anzalone, Jordan L. Doman, Gregory A. Newby, Dian Yang, Beverly Mok, Joseph M. Replogle, Albert Xu, Tyler A. Sisley, Jonathan S. Weissman, Britt Adamson & David R. Liu

doi : 10.1038/s41587-021-00938-z

Nature Biotechnology volume 39, pages1414–1425 (2021)

Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638?genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R?=?0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546?disease-related transversion single-nucleotide variants (SNVs) with?>90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE–single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.

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Adenine base editor engineering reduces editing of bystander cytosines

You Kyeong Jeong, SeokHoon Lee, Gue-Ho Hwang, Sung-Ah Hong, Se-eun Park, Jin-Soo Kim, Jae-Sung Woo & Sangsu Bae

doi : 10.1038/s41587-021-00943-2

Nature Biotechnology volume 39, pages1426–1433 (2021)

Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.

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Measurement of histone replacement dynamics with genetically encoded exchange timers in yeast

Gilad Yaakov, Felix Jonas & Naama Barkai

doi : 10.1038/s41587-021-00959-8

Nature Biotechnology volume 39, pages1434–1443 (2021)

Histone exchange between histones carrying position-specific marks and histones bearing general marks is important for gene regulation, but understanding of histone exchange remains incomplete. To overcome the poor time resolution of conventional pulse–chase histone labeling, we present a genetically encoded histone exchange timer sensitive to the duration that two tagged histone subunits co-reside at an individual genomic locus. We apply these sensors to map genome-wide patterns of histone exchange in yeast using single samples. Comparing H3 exchange in cycling and G1-arrested cells suggests that replication-independent H3 exchange occurs at several hundred nucleosomes (<1% of all nucleosomes) per minute, with a maximal rate at histone promoters. We observed substantial differences between the two nucleosome core subcomplexes: H2A-H2B subcomplexes undergo rapid transcription-dependent replacement within coding regions, whereas H3-H4 replacement occurs predominantly within promoter nucleosomes, in association with gene activation or repression. Our timers allow the in vivo study of histone exchange dynamics with minute time scale resolution.

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Prediction of drug efficacy from transcriptional profiles with deep learning

Jie Zhu, Jingxiang Wang, Xin Wang, Mingjing Gao, Bingbing Guo, Miaomiao Gao, Jiarui Liu, Yanqiu Yu, Liang Wang, Weikaixin Kong, Yongpan An, Zurui Liu, Xinpei Sun, Zhuo Huang, Hong Zhou, Ning Zhang, Ruimao Zheng & Zhengwei Xie

doi : 10.1038/s41587-021-00946-z

Nature Biotechnology volume 39, pages1444–1452 (2021)

Drug discovery focused on target proteins has been a successful strategy, but many diseases and biological processes lack obvious targets to enable such approaches. Here, to overcome this challenge, we describe a deep learning–based efficacy prediction system (DLEPS) that identifies drug candidates using a change in the gene expression profile in the diseased state as input. DLEPS was trained using chemically induced changes in transcriptional profiles from the L1000 project. We found that the changes in transcriptional profiles for previously unexamined molecules were predicted with a Pearson correlation coefficient of 0.74. We examined three disorders and experimentally tested the top drug candidates in mouse disease models. Validation showed that perillen, chikusetsusaponin IV and trametinib confer disease-relevant impacts against obesity, hyperuricemia and nonalcoholic steatohepatitis, respectively. DLEPS can generate insights into pathogenic mechanisms, and we demonstrate that the MEK–ERK signaling pathway is a target for developing agents against nonalcoholic steatohepatitis. Our findings suggest that DLEPS is an effective tool for drug repurposing and discovery.

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The RNA Atlas expands the catalog of human non-coding RNAs

Lucia Lorenzi, Hua-Sheng Chiu, Francisco Avila Cobos, Stephen Gross, Pieter-Jan Volders, Robrecht Cannoodt, Justine Nuytens, Katrien Vanderheyden, Jasper Anckaert, Steve Lefever, Aidan P. Tay, Eric J. de Bony, Wim Trypsteen, Fien Gysens, Marieke Vromman, Tine Goovaerts, Thomas Birkballe Hansen, Scott Kuersten, Nele Nijs, Tom Taghon, Karim Vermaelen, Ken R. Bracke, Yvan Saeys, Tim De Meyer, Nandan P. Deshpande, Govardhan Anande, Ting-Wen Chen, Marc R. Wilkins, Ashwin Unnikrishnan, Katleen De Preter, Jørgen Kjems, Jan Koster, Gary P. Schroth, Jo Vandesompele, Pavel Sumazin & Pieter Mestdagh

doi : 10.1038/s41587-021-00936-1

Nature Biotechnology volume 39, pages1453–1465 (2021)

Existing compendia of non-coding RNA (ncRNA) are incomplete, in part because they are derived almost exclusively from small and polyadenylated RNAs. Here we present a more comprehensive atlas of the human transcriptome, which includes small and polyA RNA as well as total RNA from 300 human tissues and cell lines. We report thousands of previously uncharacterized RNAs, increasing the number of documented ncRNAs by approximately 8%. To infer functional regulation by known and newly characterized ncRNAs, we exploited pre-mRNA abundance estimates from total RNA sequencing, revealing 316 microRNAs and 3,310 long non-coding RNAs with multiple lines of evidence for roles in regulating protein-coding genes and pathways. Our study both refines and expands the current catalog of human ncRNAs and their regulatory interactions. All data, analyses and results are available for download and interrogation in the R2 web portal, serving as a basis for future exploration of RNA biology and function.

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Author Correction: Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study

Jonathan Foox, Scott W. Tighe, Charles M. Nicolet, Justin M. Zook, Marta Byrska-Bishop, Wayne E. Clarke, Michael M. Khayat, Medhat Mahmoud, Phoebe K. Laaguiby, Zachary T. Herbert, Derek Warner, George S. Grills, Jin Jen, Shawn Levy, Jenny Xiang, Alicia Alonso, Xia Zhao, Wenwei Zhang, Fei Teng, Yonggang Zhao, Haorong Lu, Gary P. Schroth, Giuseppe Narzisi, William Farmerie, Fritz J. Sedlazeck, Don A. Baldwin & Christopher E. Mason

doi : 10.1038/s41587-021-01122-z

Nature Biotechnology volume 39, page1466 (2021)

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Author Correction: An injectable bone marrow–like scaffold enhances T cell immunity after hematopoietic stem cell transplantation

Nisarg J. Shah, Angelo S. Mao, Ting-Yu Shih, Matthew D. Kerr, Azeem Sharda, Theresa M. Raimondo, James C. Weaver, Vladimir D. Vrbanac, Maud Deruaz, Andrew M. Tager, David J. Mooney & David T. Scadden

doi : 10.1038/s41587-021-01081-5

Nature Biotechnology volume 39, page1466 (2021)

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