Publications by authors named "George M Church"

391 Publications

Benchmarking evolutionary tinkering underlying human-viral molecular mimicry shows multiple host pulmonary-arterial peptides mimicked by SARS-CoV-2.

Cell Death Discov 2020 Oct 2;6(1):96. Epub 2020 Oct 2.

nference, Cambridge, MA, USA.

The hand of molecular mimicry in shaping SARS-CoV-2 evolution and immune evasion remains to be deciphered. Here, we report 33 distinct 8-mer/9-mer peptides that are identical between SARS-CoV-2 and the human reference proteome. We benchmark this observation against other viral-human 8-mer/9-mer peptide identity, which suggests generally similar extents of molecular mimicry for SARS-CoV-2 and many other human viruses. Interestingly, 20 novel human peptides mimicked by SARS-CoV-2 have not been observed in any previous coronavirus strains (HCoV, SARS-CoV, and MERS). Furthermore, four of the human 8-mer/9-mer peptides mimicked by SARS-CoV-2 map onto HLA-B*40:01, HLA-B*40:02, and HLA-B*35:01 binding peptides from human PAM, ANXA7, PGD, and ALOX5AP proteins. This mimicry of multiple human proteins by SARS-CoV-2 is made salient by single-cell RNA-seq (scRNA-seq) analysis that shows the targeted genes significantly expressed in human lungs and arteries; tissues implicated in COVID-19 pathogenesis. Finally, HLA-A*03 restricted 8-mer peptides are found to be shared broadly by human and coronaviridae helicases in functional hotspots, with potential implications for nucleic acid unwinding upon initial infection. This study presents the first scan of human peptide mimicry by SARS-CoV-2, and via its benchmarking against human-viral mimicry more broadly, presents a computational framework for follow-up studies to assay how evolutionary tinkering may relate to zoonosis and herd immunity.
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http://dx.doi.org/10.1038/s41420-020-00321-yDOI Listing
October 2020

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

Microbiol Spectr 2021 Sep 15:e0031321. Epub 2021 Sep 15.

Department of Genetics, Harvard Medical Schoolgrid.471403.5, Boston, Massachusetts, USA.

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.
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http://dx.doi.org/10.1128/Spectrum.00313-21DOI Listing
September 2021

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

Nat Commun 2021 09 14;12(1):5429. Epub 2021 Sep 14.

Department of Genetics, Harvard Medical School, Boston, MA, USA.

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, here we report 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.
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http://dx.doi.org/10.1038/s41467-021-25691-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8440579PMC
September 2021

Cell therapy strategies for COVID-19: Current approaches and potential applications.

Sci Adv 2021 Aug 11;7(33). Epub 2021 Aug 11.

GC Therapeutics Inc., Cambridge, MA 02139, USA.

Coronavirus disease 2019 (COVID-19) continues to burden society worldwide. Despite most patients having a mild course, severe presentations have limited treatment options. COVID-19 manifestations extend beyond the lungs and may affect the cardiovascular, nervous, and other organ systems. Current treatments are nonspecific and do not address potential long-term consequences such as pulmonary fibrosis, demyelination, and ischemic organ damage. Cell therapies offer great potential in treating severe COVID-19 presentations due to their customizability and regenerative function. This review summarizes COVID-19 pathogenesis, respective areas where cell therapies have potential, and the ongoing 89 cell therapy trials in COVID-19 as of 1 January 2021.
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http://dx.doi.org/10.1126/sciadv.abg5995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357240PMC
August 2021

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells.

Sci Adv 2021 Jul 2;7(27). Epub 2021 Jul 2.

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA.

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 synthetic auxotrophy previously prevented from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.
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http://dx.doi.org/10.1126/sciadv.abf5851DOI Listing
July 2021

Deep representation learning improves prediction of LacI-mediated transcriptional repression.

Proc Natl Acad Sci U S A 2021 Jul;118(27)

Department of Genetics, Harvard Medical School, Boston, MA 02115.

Recent progress in DNA synthesis and sequencing technology has enabled systematic studies of protein function at a massive scale. We explore a deep mutational scanning study that measured the transcriptional repression function of 43,669 variants of the LacI protein. We analyze structural and evolutionary aspects that relate to how the function of this protein is maintained, including an in-depth look at the C-terminal domain. We develop a deep neural network to predict transcriptional repression mediated by the lac repressor of using experimental measurements of variant function. When measured across 10 separate training and validation splits using 5,009 single mutations of the lac repressor, our best-performing model achieved a median Pearson correlation of 0.79, exceeding any previous model. We demonstrate that deep representation learning approaches, first trained in an unsupervised manner across millions of diverse proteins, can be fine-tuned in a supervised fashion using lac repressor experimental datasets to more effectively predict a variant's effect on repression. These findings suggest a deep representation learning model may improve the prediction of other important properties of proteins.
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http://dx.doi.org/10.1073/pnas.2022838118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271634PMC
July 2021

L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.

Nat Methods 2021 06 3;18(6):631-634. Epub 2021 Jun 3.

Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA.

L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.
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http://dx.doi.org/10.1038/s41592-021-01174-8DOI Listing
June 2021

Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning.

Nat Commun 2021 05 28;12(1):3238. Epub 2021 May 28.

Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, Qingdao, China.

The design of CRISPR gRNAs requires accurate on-target efficiency predictions, which demand high-quality gRNA activity data and efficient modeling. To advance, we here report on the generation of on-target gRNA activity data for 10,592 SpCas9 gRNAs. Integrating these with complementary published data, we train a deep learning model, CRISPRon, on 23,902 gRNAs. Compared to existing tools, CRISPRon exhibits significantly higher prediction performances on four test datasets not overlapping with training data used for the development of these tools. Furthermore, we present an interactive gRNA design webserver based on the CRISPRon standalone software, both available via https://rth.dk/resources/crispr/ . CRISPRon advances CRISPR applications by providing more accurate gRNA efficiency predictions than the existing tools.
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http://dx.doi.org/10.1038/s41467-021-23576-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163799PMC
May 2021

High-throughput functional variant screens via in vivo production of single-stranded DNA.

Proc Natl Acad Sci U S A 2021 May;118(18)

Department of Genetics, Harvard Medical School, Boston, MA 02115.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
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http://dx.doi.org/10.1073/pnas.2018181118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106316PMC
May 2021

Low-N protein engineering with data-efficient deep learning.

Nat Methods 2021 04 7;18(4):389-396. Epub 2021 Apr 7.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

Protein engineering has enormous academic and industrial potential. However, it is limited by the lack of experimental assays that are consistent with the design goal and sufficiently high throughput to find rare, enhanced variants. Here we introduce a machine learning-guided paradigm that can use as few as 24 functionally assayed mutant sequences to build an accurate virtual fitness landscape and screen ten million sequences via in silico directed evolution. As demonstrated in two dissimilar proteins, GFP from Aequorea victoria (avGFP) and E. coli strain TEM-1 β-lactamase, top candidates from a single round are diverse and as active as engineered mutants obtained from previous high-throughput efforts. By distilling information from natural protein sequence landscapes, our model learns a latent representation of 'unnaturalness', which helps to guide search away from nonfunctional sequence neighborhoods. Subsequent low-N supervision then identifies improvements to the activity of interest. In sum, our approach enables efficient use of resource-intensive high-fidelity assays without sacrificing throughput, and helps to accelerate engineered proteins into the fermenter, field and clinic.
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http://dx.doi.org/10.1038/s41592-021-01100-yDOI Listing
April 2021

Ultrasensitive Measurement of Both SARS-CoV-2 RNA and Antibodies from Saliva.

Anal Chem 2021 04 23;93(13):5365-5370. Epub 2021 Mar 23.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States.

Tests for COVID-19 generally measure SARS-CoV-2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and antibodies from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive antibody test based on single molecule array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with antibody detection by Simoa identifies more patients as infected than either method alone. Our results demonstrate the utility of combining viral RNA and antibody testing from saliva, a single easily accessible biofluid.
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http://dx.doi.org/10.1021/acs.analchem.1c00515DOI Listing
April 2021

A computer-guided design tool to increase the efficiency of cellular conversions.

Nat Commun 2021 03 12;12(1):1659. Epub 2021 Mar 12.

Computational Biology Group, CIC bioGUNE-BRTA (Basque Research and Technology Alliance), Bizkaia Technology Park, Derio, Spain.

Human cell conversion technology has become an important tool for devising new cell transplantation therapies, generating disease models and testing gene therapies. However, while transcription factor over-expression-based methods have shown great promise in generating cell types in vitro, they often endure low conversion efficiency. In this context, great effort has been devoted to increasing the efficiency of current protocols and the development of computational approaches can be of great help in this endeavor. Here we introduce a computer-guided design tool that combines a computational framework for prioritizing more efficient combinations of instructive factors (IFs) of cellular conversions, called IRENE, with a transposon-based genomic integration system for efficient delivery. Particularly, IRENE relies on a stochastic gene regulatory network model that systematically prioritizes more efficient IFs by maximizing the agreement of the transcriptional and epigenetic landscapes between the converted and target cells. Our predictions substantially increased the efficiency of two established iPSC-differentiation protocols (natural killer cells and melanocytes) and established the first protocol for iPSC-derived mammary epithelial cells with high efficiency.
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http://dx.doi.org/10.1038/s41467-021-21801-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954801PMC
March 2021

Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses.

Nucleic Acids Res 2021 06;49(10):e58

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.
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http://dx.doi.org/10.1093/nar/gkab120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191787PMC
June 2021

Lineage barcoding in mice with homing CRISPR.

Nat Protoc 2021 04 10;16(4):2088-2108. Epub 2021 Mar 10.

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.
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http://dx.doi.org/10.1038/s41596-020-00485-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049957PMC
April 2021

Deep diversification of an AAV capsid protein by machine learning.

Nat Biotechnol 2021 06 11;39(6):691-696. Epub 2021 Feb 11.

Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA.

Modern experimental technologies can assay large numbers of biological sequences, but engineered protein libraries rarely exceed the sequence diversity of natural protein families. Machine learning (ML) models trained directly on experimental data without biophysical modeling provide one route to accessing the full potential diversity of engineered proteins. Here we apply deep learning to design highly diverse adeno-associated virus 2 (AAV2) capsid protein variants that remain viable for packaging of a DNA payload. Focusing on a 28-amino acid segment, we generated 201,426 variants of the AAV2 wild-type (WT) sequence yielding 110,689 viable engineered capsids, 57,348 of which surpass the average diversity of natural AAV serotype sequences, with 12-29 mutations across this region. Even when trained on limited data, deep neural network models accurately predict capsid viability across diverse variants. This approach unlocks vast areas of functional but previously unreachable sequence space, with many potential applications for the generation of improved viral vectors and protein therapeutics.
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http://dx.doi.org/10.1038/s41587-020-00793-4DOI Listing
June 2021

Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Sci Transl Med 2021 02;13(580)

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.
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http://dx.doi.org/10.1126/scitranslmed.abd3438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409505PMC
February 2021

Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency.

Nat Microbiol 2021 05 8;6(5):682-696. Epub 2021 Feb 8.

Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, China.

MicroRNA miR-138, which is highly expressed in neurons, represses herpes simplex virus 1 (HSV-1) lytic cycle genes by targeting viral ICP0 messenger RNA, thereby promoting viral latency in mice. We found that overexpressed miR-138 also represses lytic processes independently of ICP0 in murine and human neuronal cells; therefore, we investigated whether miR-138 has targets besides ICP0. Using genome-wide RNA sequencing/photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by short interfering RNA knockdown of candidate targets, we identified the host Oct-1 and Foxc1 messenger mRNAs as miR-138's targets, whose gene products are transcription factors important for HSV-1 replication in neuronal cells. OCT-1 has a known role in the initiation of HSV transcription. Overexpression of FOXC1, which was not known to affect HSV-1, promoted HSV-1 replication in murine neurons and ganglia. CRISPR-Cas9 knockout of FOXC1 reduced viral replication, lytic gene expression and miR-138 repression in murine neuronal cells. FOXC1 also collaborated with ICP0 to decrease heterochromatin on viral genes and compensated for the defect of an ICP0-null virus. In summary, miR-138 targets ICP0, Oct-1 and Foxc1 to repress HSV-1 lytic cycle genes and promote epigenetic gene silencing, which together enable favourable conditions for latent infection.
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http://dx.doi.org/10.1038/s41564-020-00860-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221016PMC
May 2021

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems.

Science 2021 01;371(6528)

Center for Cancer Genomics, Dana-Farber Cancer Institute, Boston, MA, USA.

Methods 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 the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the 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.
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http://dx.doi.org/10.1126/science.aax2656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900882PMC
January 2021

Delivery of biomacromolecules for therapeutic genome editing.

Adv Drug Deliv Rev 2021 01;168:1-2

Department of Genetics, Harvard Medical School, Boston, MA, USA. Electronic address:

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http://dx.doi.org/10.1016/j.addr.2020.12.013DOI Listing
January 2021

Characterizing the portability of phage-encoded homologous recombination proteins.

Nat Chem Biol 2021 04 18;17(4):394-402. Epub 2021 Jan 18.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA.

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.
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http://dx.doi.org/10.1038/s41589-020-00710-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990699PMC
April 2021

In situ genome sequencing resolves DNA sequence and structure in intact biological samples.

Science 2021 02 31;371(6532). Epub 2020 Dec 31.

Broad Institute of MIT and Harvard University, Cambridge, MA 02142, USA.

Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.
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http://dx.doi.org/10.1126/science.aay3446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962746PMC
February 2021

Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases.

ACS Nano 2021 01 28;15(1):489-502. Epub 2020 Dec 28.

Harvard Medical School, Department of Genetics, Boston, Massachusetts 02115, United States.

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ∼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.
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http://dx.doi.org/10.1021/acsnano.0c05226DOI Listing
January 2021

Transparency is key to ethical vaccine research.

Science 2020 12;370(6523):1422-1423

Rapid Deployment Vaccine Collaborative (RaDVaC), Lincoln, MA 01773, USA.

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http://dx.doi.org/10.1126/science.abf4851DOI Listing
December 2020

The biosecurity benefits of genetic engineering attribution.

Nat Commun 2020 12 8;11(1):6294. Epub 2020 Dec 8.

Center for Health Security, Johns Hopkins University, Baltimore, MD, USA.

Biology can be misused, and the risk of this causing widespread harm increases in step with the rapid march of technological progress. A key security challenge involves attribution: determining, in the wake of a human-caused biological event, who was responsible. Recent scientific developments have demonstrated a capability for detecting whether an organism involved in such an event has been genetically modified and, if modified, to infer from its genetic sequence its likely lab of origin. We believe this technique could be developed into powerful forensic tools to aid the attribution of outbreaks caused by genetically engineered pathogens, and thus protect against the potential misuse of synthetic biology.
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http://dx.doi.org/10.1038/s41467-020-19149-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722838PMC
December 2020

A machine learning toolkit for genetic engineering attribution to facilitate biosecurity.

Nat Commun 2020 12 8;11(1):6293. Epub 2020 Dec 8.

Alt. Technology Labs, Inc., Berkeley, CA, USA.

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 data can reach 70% attribution accuracy in 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.
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http://dx.doi.org/10.1038/s41467-020-19612-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722865PMC
December 2020

Chromosome-scale, haplotype-resolved assembly of human genomes.

Nat Biotechnol 2021 03 7;39(3):309-312. Epub 2020 Dec 7.

Department of Data Sciences, Dana-Farber Cancer Institute, Boston, MA, USA.

Haplotype-resolved or phased genome assembly provides a complete picture of genomes and their complex genetic variations. However, current algorithms for phased assembly either do not generate chromosome-scale phasing or require pedigree information, which limits their application. We present a method named diploid assembly (DipAsm) that uses long, accurate reads and long-range conformation data for single individuals to generate a chromosome-scale phased assembly within 1 day. Applied to four public human genomes, PGP1, HG002, NA12878 and HG00733, DipAsm produced haplotype-resolved assemblies with minimum contig length needed to cover 50% of the known genome (NG50) up to 25 Mb and phased ~99.5% of heterozygous sites at 98-99% accuracy, outperforming other approaches in terms of both contiguity and phasing completeness. We demonstrate the importance of chromosome-scale phased assemblies for the discovery of structural variants (SVs), including thousands of new transposon insertions, and of highly polymorphic and medically important regions such as the human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) regions. DipAsm will facilitate high-quality precision medicine and studies of individual haplotype variation and population diversity.
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http://dx.doi.org/10.1038/s41587-020-0711-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954703PMC
March 2021

Reprogramming to recover youthful epigenetic information and restore vision.

Nature 2020 12 2;588(7836):124-129. Epub 2020 Dec 2.

Department of Genetics, Blavatnik Institute, Paul F. Glenn Center for Biology of Aging Research, Harvard Medical School, Boston, MA, USA.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity. Changes to DNA methylation patterns over time form the basis of ageing clocks, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.
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http://dx.doi.org/10.1038/s41586-020-2975-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752134PMC
December 2020

A comprehensive library of human transcription factors for cell fate engineering.

Nat Biotechnol 2021 04 30;39(4):510-519. Epub 2020 Nov 30.

Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.
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http://dx.doi.org/10.1038/s41587-020-0742-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610615PMC
April 2021
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