Biology subjects

Church, G.

Publications and source records attributed to Church, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Cryptography in the DNA of living cells enabled by multi-site base editing

Although DNA is increasingly being adopted as a generalizable medium for information storage and transfer, reliable methods for ensuring information security remain to be addressed. In this study, we developed and validated a cryptographic encoding scheme, Genomic Sequence Encryption (GSE), to address the challenge of information confidentiality and integrity in biological substrates. GSE enables genomic information encoding that is readable only with a cryptographic key. We show that GSE can be used for cell signatures that enable the recipient of a cell line to authenticate its origin and validate if the cell line has been modified in the interim. We implement GSE through multi-site base editing and encode information through editing across >100 genomic sites in mammalian cells. We further present an enrichment step to obtain individual stem cells with more than two dozen edits across a single genome with minimal screening. This capability can be used to introduce encrypted signatures in living animals. As an encryption scheme, GSE is falsification-proof and enables secure information transfer in biological substrates.

synthetic biology↗

bioRxiv2023

A Platform for Controlled Template-Independent Enzymatic Synthesis of RNA Oligonucleotides and Therapeutics

Therapeutic RNA oligonucleotides have shown tremendous potential to manage and treat disease, yet current manufacturing methods cannot deliver on this promise. Here, we report the development and optimization of a novel, aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methodologies. Our platform is made possible by reversible terminator nucleoside triphosphates and an enzyme capable of their incorporation. We show that many common therapeutic RNA modifications are compatible with our process and demonstrate the enzymatic synthesis of natural and modified oligonucleotides in both liquid and solid phases. Our platform offers many unique advantages over chemical synthesis, including the realization of a more sustainable process to produce therapeutic RNA oligonucleotides. One-Sentence SummaryAn enzyme and novel nucleotide building blocks are used to synthesize RNA oligonucleotides template independently under aqueous conditions.

synthetic biology↗

bioRxiv2022

Reliable multiplex generation of pooled induced pluripotent stem cells for genetic testing

Inducing somatic cells into pluripotent stem cells (iPSCs) provides an excellent model for studying systems in-vitro. Understanding the impact of individual donor genetic backgrounds on reprogramming ability would allow researchers to harness these genetic differences and increase the efficiency of the reprogramming process. To better understand the genetic basis of reprogramming cells into iPSCs, we present Induction of Pluripotency from Pooled Cells (iPPC) - an efficient, scalable, and reliable reprogramming procedure. Using our deconvolution algorithm that employs low-coverage pooled sequencing and single nucleotide polymorphisms (SNPs), we estimate individual donor proportions of cell lines within large cohorts. With iPPC, we concurrently reprogrammed over one hundred donor LCLs into iPSCs and found strong correlations of individual donors reprogramming ability across multiple experiments. We note that individual donors reprogramming ability remains consistent across both same-day replicates and multiple experimental runs, and that the expression of certain immunoglobulin precursor genes (IGLV10-54, IGLV3-9, IGLV1-17, IGLV1-6, and IGLV3-1) may impact reprogramming ability. Our process enables a multiplex framework to study the reprogramming ability of different donor cells into iPSCs and also provides a reliable method along with a pooled library of donor iPSCs for downstream research and investigation of other in-vitro phenotypes.

molecular biology↗

bioRxiv2022

Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC

The ability to differentiate stem cells into human cell types is essential to define basic mechanisms and therapeutics, especially for cell types not routinely accessible by biopsies. But while engineered expression of transcription factors (TFs) identified through TF screens has been found to rapidly and efficiently produce some cell types, generation of other cell types that require complex combinations of TFs has been elusive. Here we develop an iterative, pooled single-cell TF screening method that improves the identification of effective TF combinations using the generation of human microglia-like cells as a testbed: Two iterations identified a combination of SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8 as sufficient to differentiate human iPSC into microglia-like cells in 4 days. Characterization of TF-induced microglia demonstrated molecular and functional similarity to primary microglia. We explore the use of single-cell atlas reference datasets to confirm identified TFs and how combining single-cell TF perturbation and gene expression data can enable the construction of causal gene regulatory networks. We describe what will be needed to fashion these methods into a generalized integrated pipeline, further ideas for enhancement, and possible applications.

bioengineering↗

bioRxiv2022

Strategies to Identify and Edit Improvements in Synthetic Genome Segments Episomally.

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments on low copy number vectors. However, DNA synthesis and amplification have the potential to impose mutations on the contained DNA segments that can in turn reduce or prevent viability of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to swap out seven codons to synonymous alternatives genome-wide. Ten 44,402 to 47,179 bp de novo synthesized BAC-contained DNA segments from the recoded strain were unable to complement deletion of the corresponding 33 to 61 wild type genes using a single antibiotic resistance marker. Next-Generation Sequencing was used to identify 1-7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the BAC-contained recoded segment when both the recoded and wild type copies of the mutated genes had to exist by necessity during the repair process. This strategy could be adapted to mutation identification and repair for other large-scale genome engineering projects, or for incorporation of small genetic engineering sites for quick protocol adjustments.

synthetic biology↗

bioRxiv2021

Reconstructing transcriptional histories by CRISPR acquisition of retron-based genetic barcodes

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons1, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record via simple, logical rules rather than relying on pre-trained classifiers or post-hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

synthetic biology↗

bioRxiv2021

Recording Temporal Signals with Minutes Resolution Using Enzymatic DNA Synthesis

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with sub-hour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3 ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals like Co2+, Ca2+, Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 minute over a 60-minute period. Finally, we engineer TdT to allosterically turn off in the presence of physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single step change in Ca2+ signal to within 1 minute over a 60-minute period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with minutes resolution.

synthetic biology↗

bioRxiv2021

Multiplex base editing to convert TAG into TAA codons in the human genome

Large-scale recoding has been shown to enable novel amino acids, biocontainment and viral resistance in bacteria only so far. Here we extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~ 40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and multiplex editing in mammalian cells.

synthetic biology↗

bioRxiv2021

New intranasal and injectable gene therapy for healthy life extension

As the global elderly population grows, it is socioeconomically and medically critical to have diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that adenovirus-associated virus (AAV) vector induced overexpression of certain proteins can suppress or reverse the effects of aging in animal models. Here, we sought to determine whether the high-capacity cytomegalovirus vector can be an effective and safe gene delivery method for two such-protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. This is the first report of CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Treatment significantly improved glucose tolerance, physical performance, and prevented loss of body mass and alopecia. Telomere shortening seen with aging was ameliorated by TERT, and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with increased health span.

cell biology↗

bioRxiv2021

Discovery and validation of novel human genomic safe harbor sites for gene and cell therapies

Existing approaches for the integration and expression of genes of interest in a desired human cellular context are marred by the safety concerns related to either the random nature of viral-mediated integration or unpredictable pattern of gene expression in currently employed targeted genomic integration sites. Disadvantages of these methods lead to their limited use in clinical practice, thus encouraging future research in identifying novel human genomic sites that allow for predictable and safe expression of genes of interest. We conducted a bioinformatic search followed by experimental validation of novel genomic sites and identified two that demonstrated stable expression of integrated reporter and therapeutic genes without detrimental changes to cellular transcriptome. The cell-type agnostic criteria used in our bioinformatic search suggest wide-scale applicability of our sites for engineering of a diverse range of tissues for therapeutic as well as enhancement purposes, including modified T-cells for cancer therapy and engineered skin to ameliorate inherited diseases and aging. Additionally, the stable and robust levels of gene expression from identified sites allow for their use in industry-scale biomanufacturing of desired proteins in human cells.

genomics↗

bioRxiv2020

Comparison of Extracellular Vesicle Isolation Methods from Human Biofluids using Single Molecule Arrays

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

biochemistry↗

bioRxiv2020

TBDB: A database of structurally annotated T-box riboswitch-tRNA pairs

T-box riboswitches constitute a large family of tRNA-binding leader sequences that play a central role in gene regulation in many gram-positive bacteria. Accurate inference of the tRNA binding to T-boxes is critical to predict their cis-regulatory activity. However, there is no central repository of information on the tRNA binding specificities of T-box riboswitches and de novo prediction of binding specificities requires advance knowledge of computational tools to annotate riboswitch secondary structure features. Here we present T-box annotation Database (TBDB, https://tbdb.io), an open-access database with a collection of 23,497 T-box sequences, spanning the major phyla of 3,621 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory target. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

bioinformatics↗

bioRxiv2020

Algorithms for the selection of fluorescent reporters

Molecular biologists rely on the use of fluorescent probes to take measurements of their model systems. These fluorophores fall into various classes (e.g. fluorescent dyes, fluorescent proteins, etc.), but they all share some general properties (such as excitation and emission spectra, brightness) and require similar equipment for data acquisition. Selecting an ideal set of fluorophores for a particular measurement technology or vice versa is a multidimensional problem that is difficult to solve with ad hoc methods due to the enormous solution space of possible fluorophore panels. Choosing sub-optimal fluorophore panels can result in unreliable or erroneous measurements of biochemical properties in model systems. Here, we describe a set of algorithms, implemented in an open-source software tool, for solving these problems efficiently to arrive at fluorophore panels optimized for maximal signal and minimal bleed-through.

bioinformatics↗

bioRxiv2020

Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

AbstractMethods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale. One Sentence SummaryIn situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

genomics↗

bioRxiv2020

Characterizing the portability of RecT-mediated oligonucleotide recombination

Bacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the hostlimited functionality of RecTs. We find that these proteins specifically recognize the 7 C-terminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through sequential nucleotide conversions.

synthetic biology↗

bioRxiv2020

Induced Pluripotent Stem Cell-derived CAR-Macrophage Cells with Antigen-dependent Anti-Cancer Cell Functions for Liquid and Solid Tumors

The Chimera antigen receptor (CAR)-T cell therapy has gained great success in the clinic. However, there are still major challenges for its wider applications in a variety of cancer types including lack of effectiveness due to the highly complex tumor microenvironment, and the forbiddingly high cost due to personalized manufacturing procedures. In order to overcome these hurdles, numerous efforts have been spent focusing on optimizing Chimera Antigen Receptors, engineering and improving T cell capacity, exploiting features of subsets of T cell or NK cells, or making off-the-shelf universal T cells. Here, we developed induced pluripotent stem cells (iPSCs)-derived, CAR-expressing macrophage cells (CAR-iMac). These cells showed antigen-dependent macrophage functions such as expression and secretion of cytokines, polarization toward the pro-inflammatory/anti-tumor state, and phagocytosis of tumor cells, as well as some in vivo anti-cancer cell activity for both liquid and solid tumors. This technology platform for the first time provides an unlimited source of iPSC-derived engineered CAR-macrophage cells which could be utilized to eliminate cancer cells or modulate the tumor microenvironment in liquid and solid tumor immunotherapy. One sentence summaryWe developed CAR-expressing iPSC-induced macrophage cells that have antigen-dependent phagocytosis and pro-inflammatory functions and anti-cancer cell activity for both liquid and solid tumor cells.

cell biology↗

bioRxiv2020

Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

Human induced pluripotent stem cell (h-iPSC)-derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated thirteen diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Importantly, timely activation of ETV2 was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.

cell biology↗

bioRxiv2020

SDip: A novel graph-based approach to haplotype-aware assembly based structural variant calling in targeted segmental duplications sequencing

Segmental duplications are important for understanding human diseases and evolution. The challenge to distinguish allelic and duplication sequences has hindered their phased assembly as well as characterization of structural variant calls. Here we have developed a novel graph-based approach that leverages single nucleotide differences in overlapping reads to distinguish allelic and duplication sequences information from long read accurate PacBio HiFi sequencing. These differences enable to generate allelic and duplication-specific overlaps in the graph to spell out phased assembly used for structural variant calling. We have applied our method to three public genomes: CHM13, NA12878 and HG002. Our method resolved 86% of duplicated regions fully with contig N50 up to 79 kb and produced <800 structural variant phased calls, outperforming state-of-the-part SDA method in terms of all metrics. Furthermore, we demonstrate the importance of phased assemblies and variant calls to the biologically-relevant duplicated genes such as SMN1, SRGAP2C, NPY4R and FAM72A. Our phased assemblies and accurate variant calling specifically in duplicated regions will enable the study of the evolution and adaptation of various species.

bioinformatics↗