Biology subjects

Al-Shayeb, B.

Publications and source records attributed to Al-Shayeb, B..

12 recordsLinked to original sources

bioRxiv2023

Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing

Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts in situ by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing. One-sentence summaryCRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.

genomics↗

bioRxiv2023

Borg extrachromosomal elements of methane-oxidizing archaea have conserved and expressed genetic repertoires

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding OmcZ nanowire-like electron-exporting cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.

microbiology↗

bioRxiv2023

Genome expansion by a CRISPR trimmer-integrase

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR-Cas1:Cas2 integrase is necessary but not sufficient. In some microbes, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. We show here that an elegant alternative pathway employs an internal exonuclease to select and process DNA for integration using the protospacer adjacent motif (PAM). A natural Cas1:Cas2-exonuclease fusion (trimmer-integrase) catalyzes coordinated DNA capture, trimming and integration. Five cryo-EM structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.

biochemistry↗

bioRxiv2022

Genome editing in plants using the compact editor Cas{Phi}

CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact Cas{Phi} nuclease is able to generate stably inherited gene edits in Arabidopsis, and that Cas{Phi} guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele we show that Cas{Phi} displays higher editing efficiency when the target locus is not DNA methylated, suggesting that Cas{Phi} is sensitive to chromatin environment. Importantly, two Cas{Phi} protein variants, vCas{Phi} and nCas{Phi}, both showed much higher editing efficiency relative to the wildtype Cas{Phi} enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that Cas{Phi} is highly specific. The hypercompact size, T-rich minimal PAM and wide range of working temperatures make Cas{Phi} an excellent supplement to existing plant genome editing systems. Significance StatementPlant genome engineering with CRISPR-Cas systems is frequently used in both research and agriculture. Here, we demonstrate that the hypercompact Cas{Phi}-2 nuclease is able to generate heritable gene edits in Arabidopsis. Two Cas{Phi} protein variants vCas{Phi} and nCas{Phi} increased the editing efficiency in plants. Cas{Phi} also has a wide range of working temperatures and the editing by Cas{Phi} is highly specific. We also observed that editing by Cas{Phi} is sensitive to chromatin environment. The hypercompact size, T-rich minimal PAM and wide range of working temperatures make Cas{Phi} an excellent supplement to existing plant genome editing systems.

plant biology↗

bioRxiv2021

Stop codon recoding is widespread in diverse phage lineages and has the potential to regulate translation of late stage and lytic genes

The genetic code is a highly conserved feature of life. However, some "alternative" genetic codes use reassigned stop codons to code for amino acids. Here, we survey stop codon recoding across bacteriophages (phages) in human and animal gut microbiomes. We find that stop codon recoding has evolved in diverse clades of phages predicted to infect hosts that use the standard code. We provide evidence for an evolutionary path towards recoding involving reduction in the frequency of TGA and TAG stop codons due to low GC content, followed by acquisition of suppressor tRNAs and the emergence of recoded stop codons in structural and lysis genes. In analyses of two distinct lineages of recoded virulent phages, we find that lysis-related genes are uniquely biased towards use of recoded stop codons. This convergence supports the inference that stop codon recoding is a strategy to regulate the expression of late stage genes and control lysis timing. Interestingly, we identified prophages with recoded stop codons integrated into genomes of bacteria that use standard code, and hypothesize that recoding may control the lytic-lysogenic switch. Alternative coding has evolved many times, often in closely related lineages, indicating that genetic code is plastic in bacteriophages and adaptive recoding can occur over very short evolutionary timescales.

microbiology↗

bioRxiv2021

The Chloroflexi supergroup is metabolically diverse and representatives have novel genes for non-photosynthesis based CO2 fixation

The Chloroflexi superphylum have been investigated primarily from the perspective of reductive dehalogenation of toxic compounds, anaerobic photosynthesis and wastewater treatment, but remain relatively little studied compared to their close relatives within the larger Terrabacteria group, including Cyanobacteria, Actinobacteria, and Firmicutes. Here, we conducted a detailed phylogenetic analysis of the phylum Chloroflexota, the phylogenetically proximal candidate phylum Dormibacteraeota, and a newly defined sibling phylum proposed in the current study, Eulabeiota. These groups routinely root together in phylogenomic analyses, and constitute the Chloroflexi supergroup. Chemoautotrophy is widespread in Chloroflexi. Two Form I Rubisco ancestral subtypes that both lack the small subunit are prevalent in ca. Eulabeiota and Chloroflexota, suggesting that the predominant modern pathway for CO2 fixation evolved in these groups. The single subunit Form I Rubiscos are inferred to have evolved prior to oxygenation of the Earths atmosphere and now predominantly occur in anaerobes. Prevalent in both Chloroflexota and ca. Eulabeiota are capacities related to aerobic oxidation of gases, especially CO and H2. In fact, aerobic and anaerobic CO dehydrogenases are widespread throughout every class-level lineage, whereas traits such as denitrification and reductive dehalogenation are heterogeneously distributed across the supergroup. Interestingly, some Chloroflexota have a novel clade of group 3 NiFe hydrogenases that is phylogenetically distinct from previously reported groups. Overall, the analyses underline the very high level of metabolic diversity in the Chloroflexi supergroup, suggesting the ancestral metabolic platform for this group enabled highly varied adaptation to ecosystems that appeared in the aerobic world.

microbiology↗

bioRxiv2021

Borgs are giant extrachromosomal elements with the potential to augment methane oxidation

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions 1, yet factors that modulate the activity of microorganisms performing this function remain little explored. In studying groundwater, sediments, and wetland soil where methane production and oxidation occur, we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins. Four curated, complete genomes are linear, up to ~1 Mbp in length and share genome organization, including replicore structure, long inverted terminal repeats, and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are a new type of archaeal extrachromosomal element with a distinct evolutionary origin. Gene sequence similarity, phylogeny, and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as "Borgs". We identified at least 19 different Borg types coexisting with Methanoperedens in four distinct ecosystems. Borg genes expand redox and respiratory capacity (e.g., clusters of multiheme cytochromes), ability to respond to changing environmental conditions, and likely augment Methanoperedens capacity for methane oxidation (e.g., methyl coenzyme M reductase). By this process, Borgs could play a previously unrecognized role in controlling greenhouse gas emissions.

microbiology↗

bioRxiv2021

A widely distributed genus of soil Acidobacteria genomically enriched in biosynthetic gene clusters

Bacteria of the phylum Acidobacteria are one of the most abundant bacterial across soil ecosystems, yet they are represented by comparatively few sequenced genomes, leaving gaps in our understanding of their metabolic diversity. Recently, genomes of Acidobacteria species with unusually large repertoires of biosynthetic gene clusters (BGCs) were reconstructed from grassland soil metagenomes, but the degree to which these species are widespread is still unknown. To investigate this, we augmented a dataset of publicly available Acidobacteria genomes with 46 metagenome-assembled genomes recovered from permanently saturated organic-rich soils of a vernal (spring) pool ecosystem in Northern California. We recovered high quality genomes for three novel species from Candidatus Angelobacter (a proposed subdivision 1 Acidobacterial genus), a genus that is genomically enriched in genes for specialized metabolite biosynthesis. Acidobacteria were particularly abundant in the vernal pool sediments, and a Ca. Angelobacter species was the most abundant bacterial species detected in some samples. We identified numerous diverse biosynthetic gene clusters in these genomes, and also in additional genomes from other publicly available soil metagenomes for other related Ca. Angelobacter species. Metabolic analysis indicates that Ca. Angelobacter likely are aerobes that ferment organic carbon, with potential to contribute to carbon compound turnover in soils. Using metatranscriptomics, we identified in situ expression of specialized metabolic traits for two species from this genus. In conclusion, we expand genomic sampling of the uncultivated Ca. Angelobacter, and show that they represent common and sometimes highly abundant members of dry and saturated soil communities, with a high degree of capacity for synthesis of diverse specialized metabolites.

microbiology↗

bioRxiv2020

Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1--Cas2 CRISPR integrase complex

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by targeting foreign DNA for destruction using CRISPR RNA-guided enzymes. CRISPR immunity begins with integration of foreign sequences into the host CRISPR genomic locus, followed by transcription and maturation of CRISPR RNAs. In a few CRISPR systems, the Cas1 integrase and a Cas6 nuclease are fused to a reverse transcriptase that enables viral sequence acquisition from both DNA and RNA sources. To determine how these components work together, we determined a 3.7 [A] resolution cryo-EM structure of a Cas6-RT-Cas1 protein complexed with Cas2, a subunit of the CRISPR integrase. The structure and accompanying mutagenesis experiments provide evidence of bidirectional crosstalk between the Cas1 and RT active sites and unidirectional crosstalk from Cas6 to the Cas1 and RT active sites. Together, these findings suggest regulated structural rearrangements that may coordinate the complexs different enzymatic activities.

biochemistry↗

bioRxiv2020

Petabase-scale sequence alignment catalyses viral discovery

Public databases contain a planetary collection of nucleic acid sequences, but their systematic exploration has been inhibited by a lack of efficient methods for searching this corpus, now exceeding multiple petabases and growing exponentially [1, 2]. We developed a cloud computing infrastructure, Serratus, to enable ultra-high throughput sequence alignment at the petabase scale. We searched 5.7 million biologically diverse samples (10.2 petabases) for the hallmark gene RNA dependent RNA polymerase, identifying well over 105 novel RNA viruses and thereby expanding the number of known species by roughly an order of magnitude. We characterised novel viruses related to coronaviruses and to hepatitis{delta} virus, respectively and explored their environmental reservoirs. To catalyse a new era of viral discovery, we established a free and comprehensive database of these data and tools. Expanding the known sequence diversity of viruses can reveal the evolutionary origins of emerging pathogens and improve pathogen surveillance for the anticipation and mitigation of future pandemics.

bioinformatics↗

bioRxiv2019

Clades of huge phage from across Earth’s ecosystems

Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earths ecosystems.

microbiology↗

bioRxiv2018

Candidate Phyla Radiation Roizmanbacteria from hot springs have novel, unexpectedly abundant, and potentially alternatively functioning CRISPR-Cas systems

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.

microbiology↗