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TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses.

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Title: TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses.
Authors: Faure, Guilhem, Saito, Makoto, Wilkinson, Max E., Quinones-Olvera, Natalia, Xu, Peiyu, Flam-Shepherd, Daniel, Kim, Stephanie, Reddy, Nishith, Zhu, Shiyou, Evgeniou, Lilia, Koonin, Eugene V., Macrae, Rhiannon K., Zhang, Feng
Source: Science. 5/1/2025, Vol. 388 Issue 6746, p1-19. 19p.
Subjects: RNA, DNA, Prokaryotes, Viruses, Transposons
Abstract: RNA-guided systems provide remarkable versatility, enabling diverse biological functions. Through iterative structural and sequence homology-based mining starting with a guide RNA-interaction domain of Cas9, we identified a family of RNA-guided DNA-targeting proteins in phage and parasitic bacteria. Each system consists of a tandem interspaced guide RNA (TIGR) array and a TIGR-associated (Tas) protein containing a nucleolar protein (Nop) domain, sometimes fused to HNH (TasH)– or RuvC (TasR)–nuclease domains. We show that TIGR arrays are processed into 36-nucleotide RNAs (tigRNAs) that direct sequence-specific DNA binding through a tandem-spacer targeting mechanism. TasR can be reprogrammed for precise DNA cleavage, including in human cells. The structure of TasR reveals striking similarities to box C/D small nucleolar ribonucleoproteins and IS110 RNA-guided transposases, providing insights into the evolution of diverse RNA-guided systems. Editor's summary: RNA-guided systems use base pairing between RNA and complementary nucleic acids to achieve precision targeting, a property that has been harnessed to create gene-editing tools. Faure et al. describe a family of RNA-guided systems called TIGRs that use a distinctive guide for DNA recognition and cleavage. The authors structurally and biochemically characterized these systems, revealing similarities to certain small nucleolar ribonucleoproteins, and demonstrated the potential to use one of them, TIGR-TasR, for gene editing. This discovery bridges gaps in our understanding of how RNA-based targeting mechanisms emerged across life, shedding light on the origins and diversity of these systems. —Di Jiang INTRODUCTION: RNA-guided systems allow single proteins (or complexes of proteins) to interact with many different target nucleic acid sequences by varying the portion of the RNA guide that is complementary to the target. In nature, RNA-guided systems have many functions, including prokaryotic adaptive immunity against invading mobile genetic elements (provided by CRISPR systems), gene regulation (provided by various classes of small RNAs), and nucleic acid modification [provided by small nucleolar RNAs (snoRNAs)]. Many of these systems, particularly CRISPR, have been developed into biotechnologies for modifying and modulating the genome and transcriptome and are being used as therapeutics. RATIONALE: Several new RNA-guided systems have been discovered on the basis of their evolutionary relationship to CRISPR systems. However, many RNA-guided systems remain undiscovered owing to limitations in evolutionary-based searches. To address this, we combined structural mining, sequence profiling, and large-language model clustering techniques to identify RNA-guided systems unrelated to CRISPR. RESULTS: Starting from the RNA binding domain in Streptococcus pyogenes Cas9 (SpCas9), we used comparative structural analysis to search for similar regions. This approach identified the nucleolar protein (Nop) domain, a conserved RNA binding fold present in IS110 recombinases, box C/D small nucleolar ribonucleoproteins (snoRNPs), and Prp31 splicing factors. Further mining uncovered a family of RNA-guided DNA-targeting systems in bacteriophages, archaeal viruses, and parasitic bacteria, which we named tandem interspaced guide RNA (TIGR)–TIGR-associated (Tas) systems. TIGR-Tas systems are modular both in their guide and effector. The guides, called tigRNAs, are encoded in arrays that contain two spacers (A and B) arranged in tandem and separated either by conserved sequences or secondary structures. Tas proteins all share a Nop domain, but some Tas proteins contain a nuclease domain, whereas others are associated with potential effector proteins. Using cryo–electron microscopy, we resolved the structure of a RuvC nuclease–containing Tas protein, TasR, from Thermoproteota archaeon isolate LB_CRA_1. The structure shows that the RuvC domain is largely separate from the core Nop domain, suggesting that it may be replaced with other functional domains in other systems. TasR cleaves DNA by using a distinctive targeting mechanism, wherein the spacers of tigRNAs specify the target by acting in tandem, with spacer A pairing to one strand of the target and spacer B pairing to the other strand. We also find striking similarities in TIGR-Tas systems to box C/D snoRNPs and IS110 RNA-guided transposases, suggesting an evolutionary connection among RNA-guided mechanisms across different domains of life. CONCLUSION: Their compact and modular structure, tandem guide RNA, and lack of target motif constraints distinguish TIGR-Tas from other RNA-guided systems and may allow for more flexible and precise genome-editing applications. We demonstrate that TasR can edit targeted loci in the human genome, highlighting its potential for further biotechnological development. Although we do not yet know the biological function of TIGR-Tas systems, they may play roles in viral defense or competition, genome regulation, or as-yet-undiscovered processes, and their arrays raise the possibility of an RNA-based memory system. TIGR-Tas systems are modular RNA-guided systems.: Comparison of the properties of CRISPR-Cas9 and TIGR-Tas RNA-guided systems. [ABSTRACT FROM AUTHOR]
Copyright of Science is the property of American Association for the Advancement of Science and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Data: TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses.
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  Data: <searchLink fieldCode="AR" term="%22Faure%2C+Guilhem%22">Faure, Guilhem</searchLink><br /><searchLink fieldCode="AR" term="%22Saito%2C+Makoto%22">Saito, Makoto</searchLink><br /><searchLink fieldCode="AR" term="%22Wilkinson%2C+Max+E%2E%22">Wilkinson, Max E.</searchLink><br /><searchLink fieldCode="AR" term="%22Quinones-Olvera%2C+Natalia%22">Quinones-Olvera, Natalia</searchLink><br /><searchLink fieldCode="AR" term="%22Xu%2C+Peiyu%22">Xu, Peiyu</searchLink><br /><searchLink fieldCode="AR" term="%22Flam-Shepherd%2C+Daniel%22">Flam-Shepherd, Daniel</searchLink><br /><searchLink fieldCode="AR" term="%22Kim%2C+Stephanie%22">Kim, Stephanie</searchLink><br /><searchLink fieldCode="AR" term="%22Reddy%2C+Nishith%22">Reddy, Nishith</searchLink><br /><searchLink fieldCode="AR" term="%22Zhu%2C+Shiyou%22">Zhu, Shiyou</searchLink><br /><searchLink fieldCode="AR" term="%22Evgeniou%2C+Lilia%22">Evgeniou, Lilia</searchLink><br /><searchLink fieldCode="AR" term="%22Koonin%2C+Eugene+V%2E%22">Koonin, Eugene V.</searchLink><br /><searchLink fieldCode="AR" term="%22Macrae%2C+Rhiannon+K%2E%22">Macrae, Rhiannon K.</searchLink><br /><searchLink fieldCode="AR" term="%22Zhang%2C+Feng%22">Zhang, Feng</searchLink>
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  Data: <searchLink fieldCode="JN" term="%22Science%22">Science</searchLink>. 5/1/2025, Vol. 388 Issue 6746, p1-19. 19p.
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  Label: Abstract
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  Data: RNA-guided systems provide remarkable versatility, enabling diverse biological functions. Through iterative structural and sequence homology-based mining starting with a guide RNA-interaction domain of Cas9, we identified a family of RNA-guided DNA-targeting proteins in phage and parasitic bacteria. Each system consists of a tandem interspaced guide RNA (TIGR) array and a TIGR-associated (Tas) protein containing a nucleolar protein (Nop) domain, sometimes fused to HNH (TasH)– or RuvC (TasR)–nuclease domains. We show that TIGR arrays are processed into 36-nucleotide RNAs (tigRNAs) that direct sequence-specific DNA binding through a tandem-spacer targeting mechanism. TasR can be reprogrammed for precise DNA cleavage, including in human cells. The structure of TasR reveals striking similarities to box C/D small nucleolar ribonucleoproteins and IS110 RNA-guided transposases, providing insights into the evolution of diverse RNA-guided systems. Editor's summary: RNA-guided systems use base pairing between RNA and complementary nucleic acids to achieve precision targeting, a property that has been harnessed to create gene-editing tools. Faure et al. describe a family of RNA-guided systems called TIGRs that use a distinctive guide for DNA recognition and cleavage. The authors structurally and biochemically characterized these systems, revealing similarities to certain small nucleolar ribonucleoproteins, and demonstrated the potential to use one of them, TIGR-TasR, for gene editing. This discovery bridges gaps in our understanding of how RNA-based targeting mechanisms emerged across life, shedding light on the origins and diversity of these systems. —Di Jiang INTRODUCTION: RNA-guided systems allow single proteins (or complexes of proteins) to interact with many different target nucleic acid sequences by varying the portion of the RNA guide that is complementary to the target. In nature, RNA-guided systems have many functions, including prokaryotic adaptive immunity against invading mobile genetic elements (provided by CRISPR systems), gene regulation (provided by various classes of small RNAs), and nucleic acid modification [provided by small nucleolar RNAs (snoRNAs)]. Many of these systems, particularly CRISPR, have been developed into biotechnologies for modifying and modulating the genome and transcriptome and are being used as therapeutics. RATIONALE: Several new RNA-guided systems have been discovered on the basis of their evolutionary relationship to CRISPR systems. However, many RNA-guided systems remain undiscovered owing to limitations in evolutionary-based searches. To address this, we combined structural mining, sequence profiling, and large-language model clustering techniques to identify RNA-guided systems unrelated to CRISPR. RESULTS: Starting from the RNA binding domain in Streptococcus pyogenes Cas9 (SpCas9), we used comparative structural analysis to search for similar regions. This approach identified the nucleolar protein (Nop) domain, a conserved RNA binding fold present in IS110 recombinases, box C/D small nucleolar ribonucleoproteins (snoRNPs), and Prp31 splicing factors. Further mining uncovered a family of RNA-guided DNA-targeting systems in bacteriophages, archaeal viruses, and parasitic bacteria, which we named tandem interspaced guide RNA (TIGR)–TIGR-associated (Tas) systems. TIGR-Tas systems are modular both in their guide and effector. The guides, called tigRNAs, are encoded in arrays that contain two spacers (A and B) arranged in tandem and separated either by conserved sequences or secondary structures. Tas proteins all share a Nop domain, but some Tas proteins contain a nuclease domain, whereas others are associated with potential effector proteins. Using cryo–electron microscopy, we resolved the structure of a RuvC nuclease–containing Tas protein, TasR, from Thermoproteota archaeon isolate LB_CRA_1. The structure shows that the RuvC domain is largely separate from the core Nop domain, suggesting that it may be replaced with other functional domains in other systems. TasR cleaves DNA by using a distinctive targeting mechanism, wherein the spacers of tigRNAs specify the target by acting in tandem, with spacer A pairing to one strand of the target and spacer B pairing to the other strand. We also find striking similarities in TIGR-Tas systems to box C/D snoRNPs and IS110 RNA-guided transposases, suggesting an evolutionary connection among RNA-guided mechanisms across different domains of life. CONCLUSION: Their compact and modular structure, tandem guide RNA, and lack of target motif constraints distinguish TIGR-Tas from other RNA-guided systems and may allow for more flexible and precise genome-editing applications. We demonstrate that TasR can edit targeted loci in the human genome, highlighting its potential for further biotechnological development. Although we do not yet know the biological function of TIGR-Tas systems, they may play roles in viral defense or competition, genome regulation, or as-yet-undiscovered processes, and their arrays raise the possibility of an RNA-based memory system. TIGR-Tas systems are modular RNA-guided systems.: Comparison of the properties of CRISPR-Cas9 and TIGR-Tas RNA-guided systems. [ABSTRACT FROM AUTHOR]
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  Label:
  Group: Ab
  Data: <i>Copyright of Science is the property of American Association for the Advancement of Science and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Text: English
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