Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12.

The TnpB proteins are transposon-associated RNA-guided nucleases that are among the most abundant proteins encoded in bacterial and archaeal genomes, but whose functions in the transposon life cycle remain unknown. TnpB appears to be the evolutionary ancestor of Cas12, the effector nuclease of type V CRISPR-Cas systems. We performed a comprehensive census of TnpBs in archaeal and bacterial genomes and constructed a phylogenetic tree on which we mapped various features of these proteins. In multiple branches of the tree, the catalytic site of the TnpB nuclease is rearranged, demonstrating structural and probably biochemical malleability of this enzyme. We identified numerous cases of apparent recruitment of TnpB for other functions of which the most common is the evolution of type V CRISPR-Cas effectors on about 50 independent occasions. In many other cases of more radical exaptation, the catalytic site of the TnpB nuclease is apparently inactivated, suggesting a regulatory function, whereas in others, the activity appears to be retained, indicating that the recruited TnpB functions as a nuclease, for example, as a toxin. These findings demonstrate remarkable evolutionary malleability of the TnpB scaffold and provide extensive opportunities for further exploration of RNA-guided biological systems as well as multiple applications.

CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics.

Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.

Generation of Anti-Mastitis Gene-Edited Dairy Goats with Enhancing Lysozyme Expression by Inflammatory Regulatory Sequence using ISDra2-TnpB System.

Gene-editing technology has become a transformative tool for the precise manipulation of biological genomes and holds great significance in the field of animal disease-resistant breeding. Mastitis, a prevalent disease in animal husbandry, imposes a substantial economic burden on the global dairy industry. In this study, a regulatory sequence gene editing breeding strategy for the successful creation of a gene-edited dairy (GED) goats with enhanced mastitis resistance using the ISDra2-TnpB system and dairy goats as the model animal is proposed. This included the targeted integration of an innate inflammatory regulatory sequence (IRS) into the promoter region of the lysozyme (LYZ) gene. Upon Escherichia Coli (E. coli) mammary gland infection, GED goats exhibited increased LYZ expression, showing robust anti-mastitis capabilities, mitigating PANoptosis activation, and alleviating blood-milk-barrier (BMB) damage. Notably, LYZ is highly expressed only in E. coli infection. This study marks the advent of anti-mastitis gene-edited animals with exogenous-free gene expression and demonstrates the feasibility of the gene-editing strategy proposed in this study. In addition, it provides a novel gene-editing blueprint for developing disease-resistant strains, focusing on disease specificity and biosafety while providing a research basis for the widespread application of the ISDra2-TnpB system.

Genome editing in plants using the TnpB transposase system.

The widely used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is thought to have evolved from IS200/IS605 transposons. TnpB proteins, encoded by one type of IS200/IS605 transposon, are considered to be the evolutionary ancestors of Cas12 nucleases, which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells. TnpB nucleases, which are smaller than Cas nucleases, have been engineered for use in genome editing in animal systems, but the feasibility of this approach in plants remained unknown. Here, we obtained stably transformed genome-edited mutants in rice (Oryza sativa) by adapting three recently identified TnpB genome editing vectors, encoding distinct TnpB nucleases (ISAam1, ISDra2, and ISYmu1), for use in plants, demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes. ISDra2 and ISYmu1 precisely edited their target sequences, with no off-target mutations detected, showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants. Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00172-6.

Genome editing using CRISPR, CAST, and Fanzor systems.

Genetic engineering technologies are essential not only for basic science but also for generating animal models for therapeutic applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, derived from adapted prokaryotic immune responses, has led to unprecedented advancements in the field of genome editing because of its ability to precisely target and edit genes in a guide RNA-dependent manner. The discovery of various types of CRISPR-Cas systems, such as CRISPR-associated transposons (CASTs), has resulted in the development of novel genome editing tools. Recently, research has expanded to systems associated with obligate mobile element guided activity (OMEGA) RNAs, including ancestral CRISPR-Cas and eukaryotic Fanzor systems, which are expected to complement the conventional CRISPR-Cas systems. In this review, we briefly introduce the features of various CRISPR-Cas systems and their application in diverse animal models.

Phylogenetic Relationships among TnpB-Containing Mobile Elements in Six Bacterial Species.

Some families of mobile elements in bacterial genomes encode not only a transposase but also an accessory TnpB gene. This gene has been shown to encode an RNA-guided DNA endonuclease, co-evolving with Y1 transposase and serine recombinase in mobile elements IS605 and IS607. In this paper, we reveal the evolutionary relationships among TnpB-containing mobile elements (TCMEs) in well-assembled genomes of six bacterial species: Bacillus cereus, Clostridioides difficile, Deinococcus radiodurans, Escherichia coli, Helicobacter pylori and Salmonella enterica. In total, 9996 TCMEs were identified in 4594 genomes. They belonged to 39 different insertion sequences (ISs). Based on their genetic structures and sequence identities, the 39 TCMEs were classified into three main groups and six subgroups. According to our phylogenetic analysis, TnpBs include two main branches (TnpB-A and TnpB-B) and two minor branches (TnpB-C and TnpB-D). The key TnpB motifs and the associated Y1 and serine recombinases were highly conserved across species, even though their overall sequence identities were low. Substantial variation was observed for the rate of invasion across bacterial species and strains. Over 80% of the genomes of B. cereus, C. difficile, D. radiodurans and E. coli contained TCMEs; however, only 64% of the genomes of H. pylori and 44% of S. enterica genomes contained TCMEs. IS605 showed the largest rate of invasion in these species, while IS607 and IS1341 had a relatively narrow distribution. Co-invasions of IS605, IS607 and IS1341 elements were observed in various genomes. The largest average copy number was observed for IS605b elements in C. difficile. The average copy numbers of most other TCMEs were smaller than four. Our findings have important implications for understanding the co-evolution of TnpB-containing mobile elements and their biological roles in host genome evolution.

Fanzor: a compact programmable RNA-guided endonuclease from eukaryotes.

The IS200/605 transposons in prokaryotes are known to harbor programmable endonucleases. Despite carrying their own transposable elements, no such effector has been characterized in eukaryotes. Saito et al. recently reported compact and programmable RNA-guided eukaryotic endonucleases, called Fanzors, that can induce targeted genetic modifications, thus expanding the genome-editing toolbox.

Estudo dos marcadores de virulência tnpA, tnpB, cagM do helicobacter pylori e sua associaçãocom afecções gástricas em Fortaleza, Brasil

O Helicobacter pylorié uma bactéria Gram-negativa que coloniza mais da metade da população mundial; a infecção por esta bactéria está associada adiversas afecções gástricas, entre elas: gastrites, úlceras pépticas ecâncer gástrico. O grau de lesões e patogenicidade é muito dependente da diversidade gênica da bactéria. O objetivo do presente estudo foi avaliar os fatores de virulência tnpA, tnpBe cagMdoH. pyloriem pacientes com gastrite, úlcera péptica e câncer gástrico. Agenotipagem das cepas de H. pylorioriundas de biópsias gástricas foi realizada pela técnica de reação em cadeia de polimerase.Foram analisados 147 pacientes, os quais 50 eram portadores de gastrite, 51 de úlceras péptica e 46 de câncer gástrico; os quais 72 eram do gênero masculino e 75 do feminino com média de idade e desvio padrão de 54,18±14,24anos.A população estudada foi dividida em duas faixas etárias, abaixo e acima de 45 anos; a qual 72,8% estavam acima de 45 anos de idade.A frequência dos genótipos estudados foi: 70(47,6%) cepas tnpA; 03(2,0%) tnpB; 19(12,9%) cagM.O gene tnpAfoi o mais presente no gênero masculino nas duas faixas etárias estudadas; no entanto, sem associação estatística do gene com tais variáveis. O gene tnpAapresentou uma associação negativa com o câncer gástrico; enquanto apresentou associação significativa com aúlcera duodenal (p= 0,002). O gene tnpB foi o de menor prevalência enão obteve nenhuma associação significante. O gene cagM foi o segundo mais prevalente e apresentou associação significativa com úlcera duodenal (p= 0,024). Concluiu-se que os genes tnpA, cagM estão correlacionados com o risco maior de desenvolver úlceras pépticas; sugerindo que tais genes são bons candidatos a serem marcadores genéticos do H. pylori para a úlcera péptica nestes pacientes de Fortaleza.

Helicobacter pyloriis a Gram-negative strainthat colonizes more than half the world's population; Infection by this strainis associated with variousgastric diseases, including: gastritis, peptic ulcers and gastric cancer. The degree of injury and pathogenicity is very dependent on the genetic diversity of the strian. The aim of this study was to evaluate the virulence factors tnpA, tnpBand cagMof H. pyloriin patients with gastritis, peptic ulcer and gastric cancer. Genotyping of H. pyloristrains arising from gastric biopsies was performed by the polymerase chain reaction technique. Were analyzed 147 patients, of which 50 had gastritis, 51 peptic ulcers and 46 gastric cancer; which 72 were male and 75 female, mean age and standard deviation of 54.18 ±14.24 years. The study population was divided into two age groups below and above 45 years; which 72.8% were above 45 years of age. The frequency of genotypes was: 70 (47.6%) tnpAstrains; 03 (2.0%) tnpB; 19 (12.9%) cagM. The tnpAgene was more prevalent in males in both age groups; however, no statistical association of the gene with such variables. The tnpAgene showed a negative association with gastric cancer; while significantly associated with duodenal ulcer (p= 0.002). The tnpBgene had the lowest prevalence and got no significant association. The cagMgene was the most prevalent second and showed a significant association with duodenal ulcer (p= 0.024). It was concluded that the tnpAgenes, cagMare correlated with increased risk of developing peptic ulcers; suggesting that these genes are good candidates for genetic markers of H. pyloriin peptic ulcer disease in these patients Fortaleza.