Ontology highlight
ABSTRACT:
PROVIDER: PRJNA991924 | ENA |
REPOSITORIES: ENA
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Project description:RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12. In eukaryotes and their viruses, TnpB homologs occur as two distinct types, Fanzor1 and Fanzor2. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, revealing that a clade of IS607 TnpBs with unusual active site arrangement found primarily in cyanobacteria likely gave rise to both types of Fanzors. The widespread nature of Fanzors imply that the properties of this particular group of IS607 TnpBs were particularly suited to adaptation and evolution in eukaryotes and their viruses. Biochemical analysis of a prokaryotic IS607 TnpB and virally encoded Fanzor1s revealed features that may have fostered co-evolution between TnpBs/Fanzors and their cognate transposases. These results provide insight into the evolutionary origins of a ubiquitous family of RNA-guided proteins that shows remarkable conservation across the three domains of life.
2023-11-18 | GSE246134 | GEO
Project description:Mutation effects prediction is a fundamental challenge in biotechnology and biomedicine. State-of-the-art computational methods have demonstrated the benefits of including semantically rich representations learned from protein sequences, but leave structural constraints out of reach. Here we developed Protein Mutational Effect Predictor (ProMEP), a general and multimodal deep representation learning method that simultaneously learns sequence context and structural constraints from proteins at the scale of evolution. ProMEP markedly outperforms current leading methods and enables accurate zero-shot mutational effects prediction across a variety of deep mutational scanning experiments. The application of ProMEP in the transposon-associated TnpB enzyme engineering task further demonstrates its ability for high-throughput protein space exploration. Without prior knowledge of TnpB, ProMEP accurately identifies multiple mutations that significantly improve the editing efficiency from millions of variants.
2024-03-13 | GSE261254 | GEO
Project description:Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605 elements undergo ‘peel-and-paste’ transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB-family genes that are evolutionarily related to the CRISPR-associated effectors Cas9 and Cas12. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that IscB and TnpB are essential to prevent loss of the donor IS element and potential transposon extinction as a consequence of the TnpA transposition mechanism. We first performed phylogenetic analysis of IscB/TnpB proteins and selected a family of related IS elements from Geobacillus stearothermophilus that we predicted would be mobilized by a common TnpA homolog. After reconstituting transposition using a heterologous expression system in E. coli, we found that IS elements were readily lost from the donor site due to the activity of TnpA in rejoining the flanking sequences back together upon excision. However, these IS elements also encode non-coding RNAs that guide TnpB and IscB nucleases to precisely recognize and cleave these excision products, leading either to elimination of the excision product or re-installation of the transposon through recombination. Indeed, under experimental conditions in which TnpA and TnpB-RNA complexes were co-expressed together with a genomically integrated IS element, transposon retention was significantly increased relative to conditions expressing TnpA alone. Remarkably, both TnpA and TnpB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between transposase and nuclease. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.
2023-06-01 | GSE223127 | GEO
Project description:CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ 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 CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wildtype CasΦ enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, flexible PAM and wide range of working temperatures make CasΦ an excellent addition to existing plant genome editing systems.
2022-12-14 | GSE206798 | GEO
Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.
2022-03-03 | E-MTAB-11497 | biostudies-arrayexpress
Project description:A genome-wide CRISPR screen was combined with a tdTomato reporter-based epigenetic memory assay to identify factors that erase epigenetic memory in ESC. After introducing genome wide perturbation and dCas9::KRAB-mediated epigenetic editing of the Esg1-tdTomato reporter, the trigger was released and cells that maintained the silencing sorted at FACS. Samples were collected out of sorted tdTomato negative (TOMminus) and positive (TOMplus) cells after 6 days of DOX treatment (epigenetic editing) and 3 or 7 days of DOX washout (release of the trigger), using a gating strategy to separate the bottom 2.5% negative cells (2.5%gate) and cells ranging from mildly to fully repressed (widegate).
2021-11-23 | E-MTAB-10524 | biostudies-arrayexpress
Project description:Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility, phage susceptibility, and host immunity. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.
2024-04-22 | GSE245749 | GEO