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1. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases

   https://doi.org/10.1126/science.abj6856  

   Altae-Tran, Han; Kannan, Soumya; Demircioglu, F. Esra; Oshiro, Rachel; Nety, Suchita P.; McKay, Luke J.; Dlakić, Mensur; Inskeep, William P.; Makarova, Kira S.; Macrae, Rhiannon K.; et al (October 2021, Science)

   IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies. 

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2. TnpB homologues exapted from transposons are RNA-guided transcription factors

   https://doi.org/10.1038/s41586-024-07598-4  

   Wiegand, Tanner; Hoffmann, Florian T; Walker, Matt_W G; Tang, Stephen; Richard, Egill; Le, Hoang C; Meers, Chance; Sternberg, Samuel H (July 2024, Nature)

   Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1–4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR–Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans7. 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 motility8, phage susceptibility9, and host immunity10. 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. RNA-guided transcription factors arose repeatedly via the domestication of transposon-encoded tnpB genes, representing a parallel evolutionary path to CRISPR-Cas adaptive immunity. 

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3. Template-guided selection of RNA ligands using imine-based dynamic combinatorial chemistry

   https://doi.org/10.1039/d0cc00266f  

   Umuhire Juru, Aline; Cai, Zhengguo; Jan, Adina; Hargrove, Amanda E. (March 2020, Chemical Communications)

   This study establishes the applicability of imine-based dynamic combinatorial chemistry to discover non-covalent ligands for RNA targets. We elucidate properties underlying the reactivity of arylamines and demonstrate target-guided amplification of tight binders in an amiloride-based dynamic library. 

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4. New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity

   https://doi.org/10.1093/nar/gkad1132  

   Molina Vargas, Adrian M.; Sinha, Souvik; Osborn, Raven; Arantes, Pablo R.; Patel, Amun; Dewhurst, Stephen; Hardy, Dwight J.; Cameron, Andrew; Palermo, Giulia; O’Connell, Mitchell R. (November 2023, Nucleic Acids Research)

   Abstract An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target–RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications. 

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5. An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

   https://doi.org/10.1038/s41477-021-01008-7  

   Sigman, Meredith J.; Panda, Kaushik; Kirchner, Rachel; McLain, Lauren L.; Payne, Hayden; Peasari, John Reddy; Husbands, Aman Y.; Slotkin, R. Keith; McCue, Andrea D. (November 2021, Nature Plants)

   Abstract In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing. 

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