Biology subjects

Doudna, J.

Publications and source records attributed to Doudna, J..

8 recordsLinked to original sources

bioRxiv2025

Phage-based microbiome manipulation elucidates interactions within community context

Testing microbial interaction hypotheses remains a major challenge in microbiome research. Bacteriophages have the potential to probe organism-organism interactions within complex microbiomes, yet are rarely used for this purpose. Here, we isolated nine narrow host range phages that replicate in several ecologically important Variovorax species. As Variovorax CL14 degrades the plant root-stunting hormone auxin, we used phages to eliminate V. CL14 from a rhizosphere consortium and reestablished the stunted root phenotype. We used three of the phages to deplete another Variovorax, SCN45, from complex communities to test correlation network-based hypotheses of thiamine interdependencies. Genome-resolved metagenomics revealed that specific taxa decreased in relative abundance following Variovorax depletion and could be rescued by thiamine supplementation. Thus, we confirmed thiamine production as the mechanistic basis for interdependence. These experiments lay the foundation for research that employs wildtype and engineered phages to test interaction hypotheses and for targeted microbiome manipulation.

microbiology↗

bioRxiv2024

Viral delivery of an RNA-guided genome editor for transgene-free plant germline editing

AbstractGenome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. But most viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here, we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

plant biology↗

bioRxiv2023

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1-3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4-10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

cell biology↗

bioRxiv2023

Assembly of SARS-CoV-2 ribonucleosomes by truncated N* variant of the nucleocapsid protein

The Nucleocapsid (N) protein of SARS-CoV-2 compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein SR region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N* or {Delta}(1-209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we show that {Delta}(1-209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of {Delta}(1-209) vRNPs requires the leucine-rich helix (LH) of the central disordered region, and that the LH promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to {Delta}(1-209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by SR phosphorylation.

microbiology↗

bioRxiv2021

Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression

Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13 enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.

bioengineering↗

bioRxiv2020

crispr2vec: Machine Learning Model Predicts Off-Target Cuts of CRISPR systems

1Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Cas systems have revolutionized gene editing, with applications in therapeutics, diagnostics, agriculture, and developing disease models. However, CRISPR-Cas suffers from off-target effects -- unintended genetic modifications in the genome that arise from its use. In this work, we present crispr2vec: a deep metric learning approach for embedding CRISPR single guide RNA (sgRNA) sequences and predicting off-target cuts. Given a fixed target sequence, we show that our learned embedding yields a faithful representation of potential off-targets. We present a new triplet sampling strategy specifically for CRISPR sequences that improves the quality of our embedding. We show the resulting embedding generalizes across different off-target cut detection assays. Finally, we demonstrate the superiority of our deep metric learning method in its ability to predict off-target cuts compared to previous literature in cross fold validation across different datasets for both seen and unseen sgRNAs.

synthetic biology↗

bioRxiv2017

A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors

CRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.

systems biology↗

bioRxiv2016

A compound that directly and selectively stalls PCSK9 translation

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome, which allows small molecules to specifically block translation of individual transcripts.\n\nOne Sentence SummaryA small-molecule PCSK9 inhibitor targets the human ribosome and selectively prevents PCSK9 synthesis.

systems biology↗