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The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.

Probiotics are live microorganisms which confer health benefits to the host. Lactic acid bacteria (LAB) are used as probiotics since decades. Enterococci being the member of LAB have proven probiotic strains; therefore, this study was aimed at finding out the potential probiotic candidates from the pool of locally isolated strains. For initial screening, one hundred and twenty-two strains were selected and subjected to different confirmatory and phenotypic tests to choose the best strains that have potential probiotic criteria, i.e., no potential virulence traits, antibiotic resistance, and having tolerance properties. Keeping this criterion, only eleven strains (n = 11) were selected for further assessment. All virulence traits such as production of hemolysin, gelatinase, biofilm, and DNase were performed and not found in the tested strains. The molecular assessment indicates the presence of few virulence-associated genes in Enterococcus faecalis strains with variable frequency. The phenotypic and genotypic assessments of antibiotic resistance profile indicate that the selected strain was susceptible to ten commonly used antibiotics, and there were no transferrable antibiotic resistance genes. The presence of CRISPR-Cas genes also confirmed the absence of antibiotic resistance genes. Various enterocin-producing genes like EntP, EntB, EntA, and EntQ were also identified in the selected strains which make them promising probiotic lead strains. Different tolerance assays like acid, NaCl, and gastric juice tolerance that mimic host conditions was also evaluated by providing artificial conditions. Cellular adhesion and aggregation properties like auto- and co-aggregation were also checked and their results reflect all in the favor of lead probiotic strains. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases. Methods: One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated. Results: We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p < 0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p < 0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system. Conclusions: Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones. [ABSTRACT FROM AUTHOR]

With the advent of a variety of vaccines against viral infections, there are multiple viruses that can be prevented via vaccination. However, breakthrough infections or uncovered strains can still cause vaccine-preventable viral infections (VPVIs). Therefore, timely diagnosis, treatment, and surveillance of these viruses is critical to patient care and public health. Point-of-care (POC) viral diagnostics tools have brought significant improvements in the detection and management of VPVIs. These cutting-edge technologies enable prompt and accurate results, enhancing patient care by facilitating timely treatment decisions. This review delves into the advancements in POC testing, including antigen/antibody detection and molecular assays, while focusing on their impact on the diagnosis, treatment, and surveillance of VPVIs such as mpox, viral hepatitis, influenza, flaviviruses (dengue, Zika, and yellow fever virus), and COVID-19. The role of POC tests in monitoring viral infection is crucial for tracking disease progression and managing outbreaks. Furthermore, the application of POC diagnostics has shown to be vital for public health strategies. In this review, we also highlight emerging POC technologies such as CRISPR-based diagnostics and smartphone-integrated POC devices, which have proven particularly beneficial in resource-limited settings. We underscore the importance of continued research to optimize these diagnostic tools for wider global use for mpox, viral hepatitis, influenza, dengue, and COVID-19, while also addressing current challenges related to their sensitivity, specificity, availability, efficiency, and more. [ABSTRACT FROM AUTHOR]

ABSTRACT Clostridium pasteurianum is a microorganism for production of 1,3‐propanediol (1,3‐PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR‐Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3‐PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3‐butanediol (2,3‐BDO) production of up to 0.48 g L−1, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain. [ABSTRACT FROM AUTHOR]

There is a critical need for improving animal resilience, welfare, and productivity to meet the nutritional needs of the growing global population. While selective breeding has brought about tremendous improvement in livestock genetics and improving traits, it is a relatively lengthy process to integrate beneficial alleles into the herd and it is not possible to introduce variants identified in other species. Therefore, gene editing provides researchers with a tool to rapidly overcome many of these challenges. This review highlights the advances in gene editing technology, the methods used to generate gene edited livestock, and approaches that can be used to accelerate the discovery of novel alleles linked to specific traits in vitro. Additionally, the application of organoid technology is discussed, and how that linked with gene editing technology can mimic the in vivo physiology and biological functions in vitro , providing answers to important biological questions and decreasing the number of large animals needed for research. Together, these tools will enable production agriculture to be more productive and thus better able to meet the growing worldwide demand for food. Identifying methods to improve animal health, production, and resilience is critical to create a sustainable food supply to meet global nutritional demand. Recent advancements in gene editing tools, cell culture methods, and in vitro phenotyping can accelerate the identification of variants or novel alleles that are transformative for livestock health and resilience. The use of organoids and gene editing can provide a model to link the genome to the phenome and decrease the number of animals needed for research. Image by Eun Su Jeon and the University of Missouri Molecular Cytology Core. This article belongs to the Collection: Proceedings of the Annual Conference of the International Embryo Technology Society, Fort Worth, TX, USA, 18–22 January 2025. [ABSTRACT FROM AUTHOR]

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems have revolutionized the field of genome editing by providing a precise, efficient, and versatile tool for manipulating genetic material. Initially discovered as an adaptive immune mechanism in prokaryotes, CRISPR-Cas systems have been repurposed for targeted genome editing in a wide range of organisms, the latest advancements in CRISPR-Cas technologies, their applications in cellular biology, and their transformative potential in therapeutic development. Particular attention is given to new variants of CRISPR systems, including base editors and prime editors, which expand the capabilities of genome editing beyond simple gene knockouts. The review also highlights key challenges, ethical considerations and future directions in the field. [ABSTRACT FROM AUTHOR]

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The creation of the CRISPR–Cas system has provided unprecedented opportunities in plant genome research and crop genetic improvement. In recent years, this system has been continuously improved to meet human needs through the expansion and modification of Cas proteins, the diversification of targeting locations, and the optimization of CRISPR vectors. In this review, we systematically describe the Class II Cas proteins that have been used in plants, deactivated Cas9 (dCas9) and its role in transcriptional regulation, precision editing systems, Cas9 protein variants, as well as methods and examples of CRISPR–Cas systems targeting various regions with different breadths. In addition, we outline the optimization plans based on CRISPR constructs that can overcome the pleiotropy of genes or accelerate the generation of transgene-free plants and the applications of CRISPR systems in plant breeding practices. Finally, we discuss the theory and development of "CRISPR plus", and the integrated application of existing systems in more species. [ABSTRACT FROM AUTHOR]

Creating fast, non-invasive, precise, and specific diagnostic tests is crucial for enhancing cancer treatment outcomes. Among diagnostic methods, those relying on nucleic acid detection are highly sensitive and specific. Recent developments in diagnostic technologies, particularly those leveraging Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are revolutionizing cancer detection, providing accurate and timely results. In clinical oncology, liquid biopsy has become a noninvasive and early-detectable alternative to traditional biopsies over the last two decades. Analyzing the nucleic acid content of liquid biopsy samples, which include Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA), Circulating Cell-Free RNA (cfRNA), and tumor extracellular vesicles, provides a noninvasive method for cancer detection and monitoring. In this review, we explore how the characteristics of various Cas (CRISPR-associated) enzymes have been utilized in diagnostic assays for cancer liquid biopsy and highlight their main applications of innovative approaches in monitoring, as well as early and rapid detection of cancers. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a desirable gene modification tool covering a wide area in various sectors of medicine, agriculture, and microbial biotechnology. The role of this incredible genetic engineering technology has been extensively investigated; however, it remains formidable with cargo choices, nonspecific delivery, and insertional mutagenesis. Various nanomaterials including lipid, polymeric, and inorganic are being used to deliver the CRISPR-Cas system. Progress in nanomaterials could potentially address these challenges by accelerating precision targeting, cost-effectiveness, and one-step delivery. In this review, we highlighted the advances in nanotechnology and nanomaterials as smart delivery systems for CRISPR-Cas so as to ameliorate applications for environmental remediation including biomedical research and healthcare, strategies for mitigating antimicrobial resistance, and to be used as nanofertilizers for enhancing crop growth, and reducing the environmental impact of traditional fertilizers. The timely co-evolution of nanotechnology and CRISPR technologies has contributed to smart novel nanostructure hybrids for improving the onerous tasks of environmental remediation and biological sustainability. [ABSTRACT FROM AUTHOR]

Viruses, known for causing widespread biological harm and even extinction, pose significant challenges to public health. Virus detection is crucial for accurate disease diagnosis and preventing the spread of infections. Recently, the outstanding analytical performance of CRISPR/Cas biosensors has shown great potential and they have been considered as augmenting methods for reverse-transcription polymerase chain reaction (RT-PCR), which was the gold standard for nucleic acid detection. We herein utilized Cas12a with universal CRISPR RNA (crRNA) for indiscriminate virus detection by attaching the target to a longer track strand for isothermal amplification. The amplified products contain a domain that is recognized by the Cas12a/crRNA complex, triggering the cleavage of surrounding reporters to produce signals, thereby escaping the target dependence of crRNA recognition. The proposed method allows the same crRNA to detect multiple viral nucleic acids with high sensitivity, including but not limited to SARS-CoV-2, human papillomaviruses (HPV), HCOV-NL63, HCOV-HKU1, and miRNA biomarkers. Taking SARS-CoV-2 and HPV16 pseudoviruses as examples, this method was proved as a versatile and sensitive platform for molecular diagnostic applications. [ABSTRACT FROM AUTHOR]

CRISPR–Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR–Cas-based therapies. This review examines the current landscape of CRISPR–Cas implementation in clinical trials, with data from key registries, including the Australian New Zealand Clinical Trials Registry, the Chinese Clinical Trial Register, and ClinicalTrials.gov. Emphasis is placed on the mechanism of action of tested therapies, the delivery method, and the most recent findings of each clinical trial. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system against bacteriophages. In this study, 675 sequences of Lactiplantibacillus plantarum isolates deposited in GenBank were analyzed in terms of diversity, occurrence, and evolution of the CRISPR-Cas system. This study investigated the presence, structural variations, phylogenetic relationships, and diversity of CRISPR-Cas systems in 675 L. plantarum strains. The analysis revealed that 143 strains harbor confirmed CRISPR-Cas systems, with subtype II-A being predominant. Moreover, targeting phages and plasmid diversity between the predicted systems were dissected. The results indicated that approximately 22% of the isolates with verified and complete CRISPR systems exhibited the coexistence of both subtypes II-A and I-E within their genomes. The results further showed that in subtype II-A, the length of the repeat sequence was 36 nucleotides, on average. In addition, the number of spacers in subtypes II-A and I-E varied between 1–24 and 3–16 spacers, respectively. The results also indicated that subtype II-A has nine protospacer adjacent motifs, which are 5′-CC-3′, 5′-GAA-3′, 5′-TGG-3′, 5′-CTT-3′, 5′-GGG-3′, 5′-CAT-3′, 5′-CTC-3′, 5′-CCT-3′, and 5′-CGG-3′. In addition, the identified systems displayed a potential for targeting Lactobacillus phages. The investigation of the relationship between the targeting of Lactobacillus phages by the antiphage system in L. plantarum species showed that subtype II-A had the highest diversity in targeting Lactobacillus phages than subtype I-E. In conclusion, current findings offer a perspective on the prevalence and evolution of the CRISPR-Cas system in L. plantarum , contributing novel insights to the expanding field of CRISPR-Cas systems within lactobacillus strains. This knowledge establishes a foundation for future applied studies focused on enhancing phage resistance in industrial fermentation, reducing contamination risks, and improving product quality. The identified targeting diversity may also foster advancements in phage therapy through the development of CRISPR-based antimicrobials. [ABSTRACT FROM AUTHOR]

CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system. [ABSTRACT FROM AUTHOR]

The CRISPR-Cas system has revolutionized precision genome editing and opened up enormous prospects for crop improvement and trait enhancement. This review covers recent advances related to the application of CRISPR-Cas in agricultural biotechnology, with an emphasis on its potential to contribute to solving critical issues of food security and sustainability. Global population is expected to reach 10 billion by 2050, presenting a challenge that demands new approaches to raise agricultural productivity. The review investigates the mechanisms by which CRISPRCas systems, especially Cas9 and Cas12a, facilitate precise alterations in plant genomes, leading to augmented resilience against both biological and environmental challenges. Significant accomplishments comprise increased drought resistance, enhanced disease tolerance, and improved nutritional quality in agricultural species such as wheat, maize, and soybean. However, the application of CRISPR-Cas technology encounters challenges, including regulatory hurdles and public acceptance. This paper underlines modern achievements together with future possibilities, at the same time paying much attention to the connected challenges and emphasizing the crucial role of CRISPR-Cas technology for the advancement of sustainable agriculture. Using such a powerful tool, scientists aim at creating resistant crop species that will be able to meet the needs of the growing population while providing food safety and quality. [ABSTRACT FROM AUTHOR]

The increasing global energy demand and environmental concerns have highlighted the need for sustainable and renewable energy sources. Lignocellulosic biomass (LCB), rich in cellulose, hemicellulose, and lignin, is a promising resource for biofuel production. However, the recalcitrant nature of lignin poses a significant challenge by obstructing effective LCB decomposition. This review provides a comprehensive analysis of recent advancements in genetic and metabolic engineering techniques, focusing on directed and random mutagenesis to enhance cellulase production in fungi. It explores how these techniques can overcome challenges in lignin degradation and improve LCB conversion efficiency. Lignin's high resistance to degradation, due to its complex association with cellulose and hemicelluloses, necessitates the development of advanced fungal strains through mutagenesis. Fungi, which are efficient lignin degraders, benefit from these practices to enhance enzyme production and address environmental pollution from burning LCB. The review emphasizes engineering cellulolytic fungi through mutagenesis, gene-editing, and synthetic biology, highlighting CRISPR-Cas innovations and nanoparticle-based delivery systems for precise CRISPR-Cas application. It also discusses the role of transcription factors in boosting enzyme production and the practical applications of these techniques for in-situ LCB biodegradation. Effective implementation of these advancements could foster a sustainable economy and mitigate the negative environmental impacts of current agricultural practices.Article Highlights: CRISPR-Cas technology enhances the precision and efficiency of cellulase production in fungi. Mutagenesis is key to developing fungi that break down lignin better, boosting biofuel production. Nanotechnology improves CRISPR-Cas delivery, enabling precise gene editing for sustainable bioengineering. [ABSTRACT FROM AUTHOR]

The CRISPR-Cas system, an adaptive immunity system in prokaryotes designed to combat phages and foreign nucleic acids, has evolved into a groundbreaking technology enabling gene knockout, large-scale gene insertion, base editing, and nucleic acid detection. Despite its transformative impact, the conventional CRISPR-Cas effectors face a significant hurdle—their size poses challenges in effective delivery into organisms and cells. Recognizing this limitation, the imperative arises for the development of compact and miniature gene editors to propel advancements in gene-editing-related therapies. Two strategies were accepted to develop compact genome editors: harnessing OMEGA (Obligate Mobile Element-guided Activity) systems, or engineering the existing CRISPR-Cas system. In this review, we focus on the advances in miniature genome editors based on both of these strategies. The objective is to unveil unprecedented opportunities in genome editing by embracing smaller, yet highly efficient genome editors, promising a future characterized by enhanced precision and adaptability in the genetic interventions. [ABSTRACT FROM AUTHOR]

Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts. [ABSTRACT FROM AUTHOR]

Abstract Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.