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PubMed2025

Experimental strategy for characterization of novel TnpB orthologs.

TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.

Humans↗
PubMed2019

Targeted delivery of CRISPR interference system against Fabp4 to white adipocytes ameliorates obesity, inflammation, hepatic steatosis, and insulin resistance.

Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.

3T3 Cells↗
PubMed2026

One Plasmid Is All You Need: Genome Editing in Escherichia coli Using Endogenous TnpB and Endogenous Recombination System.

Escherichia coli (E. coli) is a key workhorse of biotechnology. Commonly used CRISPR-Cas9 systems for E. coli genome editing are complex and impose metabolic stress on the host, creating demand for more streamlined strategies. Recent studies identified the IS605 transposon-associated TnpB as a programmable RNA-guided (ωRNA) DNA endonuclease, prompting us to explore whether endogenous TnpB in E. coli (EcoTnpB) could be harnessed for genome editing. Biochemical and cellular analyses demonstrated that EcoTnpB efficiently cleaves both chromosomal and plasmid DNA at custom-specified sites in a TAM-dependent manner. Interestingly, E. coli possesses an endogenous recombination machinery capable of repairing EcoTnpB-induced DNA double-strand breaks (DSBs), challenging the long-held view that bacteria lack efficient homologous recombination systems. Based on these findings, we established a single-plasmid editing system (SPEED) in which genome editing is achieved by simply providing ωRNA and a homologous recombination template. By utilizing endogenous EcoTnpB together with the host HR pathway, this system enabled inducible and seamless genome editing at multiple genomic loci in BL21 (DE3), with editing efficiencies ranging from approximately 29% to 56%. Our results demonstrate for the first time that endogenous TnpB can be harnessed for genome editing and may hold potential for broader applications, such as species-specific antimicrobial development.

Escherichia coli↗
PubMed2004

Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions.

Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.

Animals↗
PubMed2024

DNA-guided CRISPR/Cas12 for RNA targeting.

CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.

Journal Article↗
PubMed2025

DNA-guided CRISPR/Cas12 for RNA targeting.

CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12's potential for diagnostic applications and for targeted RNA modulation in cellular environments.

Journal Article↗
PubMed2005

Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans.

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.

Animals↗
PubMed2004

Involvement of the nucleolus in plant virus systemic infection.

The nucleolus is a prominent subnuclear domain and is classically regarded as the site of transcription of rRNA, processing of the precursor rRNAs and biogenesis of pre-ribosomal particles. In addition to these traditionally recognized activities, the nucleolus also participates in many other aspects of cell function. The umbravirus-encoded ORF3 protein is a multifunctional RNA-binding protein involved in long-distance RNA movement, and protection of viral RNA from RNase attack, including possibly small interfering RNA-guided RNA silencing. In addition to its presence in cytoplasmic ribonucleoprotein particles containing viral RNA, the umbraviral ORF3 protein accumulates in nuclei, preferentially targeting nucleoli. The ORF3 protein domains involved in the localization of the protein to the nucleolus were identified. Functional analysis of the mutants revealed the correlation between the ORF3 protein nucleolar localization and its ability to form the cytoplasmic ribonucleoprotein particles and transport viral RNA long distances via the phloem. Possible mechanisms of the nucleolar involvement in systemic virus infection are discussed.

Amino Acid Sequence↗
PubMed1992

Formation of guide RNA/messenger RNA chimeric molecules in vitro, the initial step of RNA editing, is dependent on an anchor sequence.

Synthetic pre-edited messenger RNA (mRNA) and guide RNA (gRNA) for the 5'-edited maxicircle-encoded ND7 cryptogene from Leishmania tarentolae formed chimeric molecules upon incubation in the presence of a mitochondrial extract. These chimeric molecules consisted of the gRNAs covalently linked to the mRNAs by short oligo(U) tails at normal editing sites in most cases. Unlike the previously reported chimeras present in steady-state kinetoplast RNA, the in vitro-synthesized chimeras showed no editing of downstream editing sites. The synthesis of chimeric RNAs required ATP and was dependent on the formation of a gRNA/mRNA anchor duplex 3' of the pre-edited region, as shown by in vitro mutagenesis of the mRNA and the gRNA. mRNA sequences 3' and 5' of the pre-edited region also affected the efficiency of the chimera-forming activity. This in vitro system may accurately represent the initial step in RNA editing.

Animals↗
PubMed2001

Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21.

RNA editing within the mitochondria of African trypanosomes is characterized by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The reaction takes place in a high molecular mass ribonucleoprotein (RNP) complex of uncertain composition. Furthermore, factors that interact with the RNP complex during the reaction are by and large unknown. Here we present evidence for an editing-related biochemical activity of the gRNA-binding protein gBP21. Using recombinant gBP21 preparations, we show that the protein stimulates the annealing of gRNAs to cognate pre-mRNAs in vitro. This represents the presumed first step of the editing reaction. Kinetic data establish an enhancement of the second order rate constant for the gRNA- pre-mRNA interaction. gBP21-mediated annealing is not exclusive for RNA editing substrates since complementary RNAs, unrelated to the editing process, can also be hybridized. The gBP21-dependent RNA annealing activity was identified in mitochondrial extracts of trypanosomes and can be inhibited by immunoprecipitation of the polypeptide. The data suggest a factor-like contribution of gBP21 to the RNA editing process by accelerating the rate of gRNA-pre-mRNA anchor formation.

Animals↗
PubMed2006

Chromosome healing by de novo telomere addition in Saccharomyces cerevisiae.

The repair of spontaneous or induced DNA damage by homologous recombination (HR) in Saccharomyces cerevisiae will suppress chromosome rearrangements. Alternative chromosome healing pathways can result in chromosomal instability. One of these pathways is de novo telomere addition where the end of a broken chromosome is stabilized by telomerase-dependent addition of telomeres at non-telomeric sites. De novo telomere addition requires the recruitment of telomerase to chromosomal targets. Subsequently, annealing of the telomerase reverse transcriptase RNA-template (guide RNA) at short regions of homology is followed by extension of the nascent 3'-end of the broken chromosome to copy a short region of the telomerase guide RNA; multiple cycles of this process yield the new telomere. Proteins including Pif1 helicase, the single-stranded DNA-binding protein Cdc13 and the Ku heterocomplex are known to participate in native telomere functions and also regulate the de novo telomere addition reaction. Studies of the sequences added at de novo telomeres have lead to a detailed description of the annealing-extension-dissociation cycles that copy the telomerase guide RNA, which can explain the heterogeneity of telomeric repeats at de novo and native telomeres in S. cerevisiae.

Chromosome Breakage↗
PubMed1996

Guide RNA-directed uridine insertion RNA editing in vitro.

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.

Animals↗
PubMed2000

Activation of guide RNA-directed editing of a cytochrome b mRNA.

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created only after the addition or deletion of specific uridines. Although in vitro systems have been valuable in characterizing the editing mechanism, only a limited number of mRNAs are accurately edited in vitro. We demonstrate here that in vitro editing of cytochrome b mRNA is inhibited by an A-U sequence present on both the 5'-untranslated sequence and on a cytochrome b guide RNA. Mutation of the sequence on the guide RNA stimulates directed editing and results in the loss of binding to at least one component within the editing extract. Mutation of the sequence on the mRNA increases the accuracy of the editing. Evidence is provided that suggests the A-U sequence interacts with the editing machinery both in vitro and in vivo.

Base Sequence↗
PubMed1997

RNA processing: pocket guides to ribosomal RNA.

The functional role of a recently identified class of small nucleolar (sno) RNAs has been elucidated: the 'box H/ACA' snoRNAs act as guide RNAs, specifying the position of evolutionarily conserved pseudouridines in ribosomal (r)RNA via an rRNA-snoRNA base-pairing interaction that forms a 'pseudouridine pocket'.

Base Composition↗
PubMed2003

Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae.

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

Animals↗
PubMed1995

Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase.

RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation.

Animals↗
PubMed1992

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

Animals↗
PubMed1995

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.

Amino Acid Sequence↗