Biology subjects

Church, G. M.

Publications and source records attributed to Church, G. M..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Improved Isolation of Extracellular Vesicles by Removal of Both Free Proteins and Lipoproteins

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly-sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan*, Norman* et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography (SEC) with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main contaminants of EV isolation in plasma and apply this approach to develop novel methods for isolating highly-pure EVs from human plasma. These methods will enable applications where high purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.

bioengineering↗

bioRxiv2022

Machine-guided cell-fate engineering

AO_SCPLOWBSTRACTC_SCPLOWThe creation of induced pluripotent stem cells (iPSCs) has enabled scientists to explore the derivation of many types of cells. While there are diverse general approaches for cell-fate engineering, one of the fastest and most efficient approaches is transcription factor (TF) over-expression. However, finding the right combination of TFs to over-express to differentiate iPSCs directly into other cell-types is a difficult task. Here were describe a machine-learning (ML) pipeline, called CellCartographer, for using chromatin accessibility data to design multiplex TF pooled-screens for cell type conversions. We validate this method by differentiating iPSCs into twelve diverse cell types at low efficiency in preliminary screens and then iteratively refining our TF combinations to achieve high efficiency differentiation for six of these cell types in < 6 days. Finally, we functionally characterized engineered iPSC-derived cytotoxic T-cells (iCytoT), regulatory T-cells (iTReg), type II astrocytes (iAstII), and hepatocytes (iHep) to validate functionally accurate differentiation.

developmental biology↗

bioRxiv2022

Expansion Sequencing of RNA Barcoded Neurons in the Mammalian Brain: Progress and Implications for Molecularly Annotated Connectomics

Mapping and molecularly annotating mammalian neural circuits is challenging due to the inability to uniquely label cells while also resolving subcellular features such as synaptic proteins or fine cellular processes. We argue that an ideal technology for connectomics would have the following characteristics: the capacity for robust distance-independent labeling, synaptic resolution, molecular interrogation, and scalable computational methods. The recent development of high-diversity cellular barcoding with RNA has provided a way to overcome the labeling limitations associated with spectral dyes, however performing all-optical circuit mapping has not been demonstrated because no method exists to image barcodes throughout cells at synaptic-resolution. Here we show ExBarSeq, an integrated method combining in situ sequencing of RNA barcodes, immunostaining, and Expansion Microscopy coupled with an end-to-end software pipeline that automatically extracts barcode identities from large imaging datasets without data processing bottlenecks. As a proof of concept, we applied ExBarSeq to thick tissue sections from mice virally infected with MAPseq viral vectors and demonstrated the extraction of 50 barcoded cells in the visual cortex as well as cell morphologies uncovered via immunostaining. The current work demonstrates high resolution multiplexing of exogenous barcodes and endogenous synaptic proteins and outlines a roadmap for molecularly annotated connectomics at a brain-wide scale.

neuroscience↗

bioRxiv2022

Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors

The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive modeling and medicine. Methods to recapitulate advanced germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described graph theory pipeline and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX whose combinatorial overexpression drives DDX4+ induced oogonia-like cell (iOLC) formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We additionally demonstrate a method of post-isolation, feeder-free expansion of DDX4+ iOLCs that shows retained cell identity in vitro. We additionally identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their broad similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.

developmental biology↗

bioRxiv2022

Swapped genetic code blocks viral infections and gene transfer

Removing cellular transfer RNAs (tRNAs), making their cognate codons unreadable, creates a genetic firewall that prevents viral replication and horizontal gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, including tRNAs, potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such horizontally transferred tRNA genes can enable viral replication in Escherichia coli cells despite the genome-wide lack of three codons and the previously essential cognate tRNAs and release factor 1. By repurposing viral tRNAs, we then develop recoded cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells completely resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. Finally, we also repurpose the third free codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature.

synthetic biology↗

bioRxiv2022

Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells form ovary-like organoids (ovaroids) when aggregated with hPGCLCs, and recapitulate key ovarian phenotypes including support of germ cell maturation, follicle formation, and steroidogenesis. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

developmental biology↗

bioRxiv2022

Efficient Modification and Preparation of Circular DNA for Expression in Cell Culture

DNA plasmids are an essential tool for delivery and expression of RNAs and proteins in cell culture experiments. The preparation of plasmids typically involves a laborious process of bacterial cloning, validation, and purification. While the expression plasmids can be designed and ordered from the contract manufacturers, the cost may be prohibitive when a large number of plasmids is required. We have developed an efficient fully synthetic method and protocol that enables the production of circularized DNA containing expression elements ready for transfection in as little as 3 hours, thereby eliminating the bacterial cloning steps. The protocol describes how to take a linear double-stranded DNA fragment and efficiently circularize and purify this DNA fragment with minimal hands-on time. As proof of the principle, we applied Circular Vector expressing engineered prime editing guide RNA (epegRNA) in cell culture, and demonstrated matching and even exceeding performance of this method as compared to guides expressed by plasmids. The method is not limited to gene editing and its speed of preparation, low cost, and ease of use will make it a useful tool in a variety of applications requiring the expression of short RNAs and proteins.

genomics↗

bioRxiv2022

Multiplex CRISPR strategy targeting viral genome for agriculture and clinical use

African Swine Fever (ASF) is a viral disease with exceptionally high lethality in domestic pigs and wild boar worldwide1, 2, without any effective vaccine or drug to prevent its spread. In this study, we established a multiplexable CRISPR-Cas-gRNA system targeting 13 genomic loci in the ASF virus genome that could prevent viral replication by cutting its genome. Furthermore, we generated pig strains expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing and demonstrated that the gene-edited pigs were more resistant to ASFV infection and less likely to spread the virus upon infection. As far as we know, our study presents the first living organism generated via germline editing to demonstrate resistance to viral infection via CRISPR-Cas. We anticipate our work to be helpful for both agricultural and biomedical applications, such as xenotransplantation.

bioengineering↗

bioRxiv2022

helixCAM: A Platform for Programmable Cellular Assembly in Bacteria and Human Cells

Interactions between cells are indispensable for creating structure and signaling. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, CAMs are natively expressed by many cells and have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering novel CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. We built rationally designed helixCAM libraries and discovered novel high-performance helixCAM pairs. High-affinity helixCAMs were then used for multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning. The helixCAMs are an expandable platform for directing complex multicellular assemblies, and we foresee its utility in tissue engineering, immunology, and developmental biology.

synthetic biology↗

bioRxiv2021

PGP1 personal genome assembly - a hybrid assembly dataset using ONT's PromethION and PacBio's HiFi sequencing

PGP1 is the first participant of Personal Genome Project. We present the PGP1s chromosome-scale genome assembly. It was constructed using 255 Gb ultra-long PromethION reads and 97 Gb short paired-end reads. For reducing base calling errors, we corrected PromethION reads using 72 Gb PacBio HiFi reads. 327 Gb Hi-C chromosomal mapping data were utilized to maximize the assemblys contiguity. PGP1s contig assembly was 3.01 Gb in length comprising of 4,234 contigs with an N50 value of 33.8 Mb. After scaffolding with Hi-C data and extensive manual curation, we obtained a chromosome-scale assembly that represents 3,880 scaffolds with an N50 value of 142 Mb. From the Merqury assessment, PGP1 assembly achieved a high QV score of Q45.45. For a gene annotation, we predicted 106,789 genes with a liftover from the Gencode 38 and an assembly of transcriptome data.

genomics↗

bioRxiv2021

Single-sequence protein structure prediction using language models from deep learning

AlphaFold2 and related systems use deep learning to predict protein structure from co-evolutionary relationships encoded in multiple sequence alignments (MSAs). Despite dramatic, recent increases in accuracy, three challenges remain: (i) prediction of orphan and rapidly evolving proteins for which an MSA cannot be generated, (ii) rapid exploration of designed structures, and (iii) understanding the rules governing spontaneous polypeptide folding in solution. Here we report development of an end-to-end differentiable recurrent geometric network (RGN) able to predict protein structure from single protein sequences without use of MSAs. This deep learning system has two novel elements: a protein language model (AminoBERT) that uses a Transformer to learn latent structural information from millions of unaligned proteins and a geometric module that compactly represents C backbone geometry. RGN2 outperforms AlphaFold2 and RoseTTAFold (as well as trRosetta) on orphan proteins and is competitive with designed sequences, while achieving up to a 106-fold reduction in compute time. These findings demonstrate the practical and theoretical strengths of protein language models relative to MSAs in structure prediction.

bioinformatics↗

bioRxiv2021

Generative AAV capsid diversification by latent interpolation

Adeno-associated virus (AAV) capsids have shown clinical promise as delivery vectors for gene therapy. However, the high prevalence of pre-existing immunity against natural capsids poses a challenge for widespread treatment. The generation of diverse capsids that are potentially more capable of immune evasion is challenging because introducing multiple mutations often breaks capsid assembly. Here we target a representative, immunologically relevant 28-amino-acid segment of the AAV2 capsid and show that a low-complexity Variational Auto-encoder (VAE) can interpolate in sequence space to produce diverse and novel capsids capable of packaging their own genomes. We first train the VAE on a 564-sample Multiple-Sequence Alignment (MSA) of dependo-parvoviruses, and then further augment this dataset by adding 22,704 samples from a deep mutational exploration (DME) on the target region. In both cases the VAE generated viable variants with many mutations, which we validated experimentally. We propose that this simple approach can be used to optimize and diversify other proteins, as well as other capsid traits of interest for gene delivery.

synthetic biology↗

bioRxiv2021

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

Bacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

synthetic biology↗

bioRxiv2020

Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications. One Sentence SummaryTo ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

synthetic biology↗

bioRxiv2020

A DNA nanoscope that identifies and precisely localizes over a hundred unique molecular features with nanometer accuracy

Techniques that can both spatially map out molecular features and discriminate many targets would be highly valued for their utility in studying fundamental nanoscale processes. In spite of decades of development, no current technique can achieve both nanoscale resolution and discriminate hundreds of targets. Here, we report the development of a novel bottom-up technology that: (a) labels a sample with DNA barcodes, (b) measures pairwise-distances between labeled sites and writes them into DNA molecules, (c) reads the pairwise-distances by sequencing and (d) robustly integrates this noisy information to reveal the geometry of the underlying sample. We demonstrate our technology on DNA origami, which are complex synthetic nanostructures. We both spatially localized and uniquely identified over a hundred densely packed unique elements, some spaced just 6 nm apart, with an average spatial localization accuracy (RMS deviation) of ~2 nm. The bottom-up, sequencing-enabled mechanism of the DNA nanoscope is fundamentally different from top-down imaging, and hence offers unique advantages in precision, throughput and accessibility.

biochemistry↗

bioRxiv2020

Attribution of genetic engineering: a practical and accurate machine learning toolkit for biosecurity

The promise of biotechnology is tempered by its potential for accidental or deliberate misuse. Reliably identifying telltale signatures characteristic to different genetic designers, termed genetic engineering attribution, would deter misuse, yet is still considered unsolved. Here, we show that recurrent neural networks trained on DNA motifs and basic phenotype can reach 70% attribution accuracy distinguishing between over 1,300 labs. To make these models usable in practice, we introduce a framework for weighing predictions against other investigative evidence using calibration, and bring our model to within 1.6% of perfect calibration. Additionally, we demonstrate that simple models can accurately predict both the nation-state-of-origin and ancestor labs, forming the foundation of an integrated attribution toolkit which should promote responsible innovation and international security alike.

synthetic biology↗

bioRxiv2020

L1CAM is not Associated with Extracellular Vesicles in Human Cerebrospinal Fluid or Plasma

Neuron-derived extracellular vesicles (NDEVs) present a tremendous opportunity to learn about the biochemistry of brain cells in living patients. L1CAM is a transmembrane protein expressed in neurons that is presumed to be found on NDEVs in human biofluids. Previous studies have used L1CAM immuno-isolation from human plasma to isolate NDEVs for neurodegenerative disease diagnostics. We developed a panel of ultrasensitive Single Molecule Array (Simoa) assays for known EV markers, as well as L1CAM, and applied it to study EVs in human plasma and cerebrospinal fluid (CSF). We fractionated plasma and CSF by size exclusion chromatography (SEC) and density gradient centrifugation (DGC) to separate EVs from free proteins. We observed that L1CAM did not elute in the EV fractions, but rather eluted in the free protein fractions. We found that L1CAM is present as a free protein in human plasma and CSF, possibly due to proteolytic cleavage and/or alternative splicing. We further demonstrate that the isoforms found in CSF and plasma are different. These data collectively establish that L1CAM in plasma is not EV associated and should therefore not be used for NDEV isolation. Importantly, the framework and tools described herein will allow for evaluation of other potential candidate markers for isolation of NDEVs.

neuroscience↗

bioRxiv2020

Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

systems biology↗