Ilya J. Finkelstein

Subtelomeres are imperfect repeats adjacent to telomeres that are transcriptionally repressed by heterochromatin. Although essential for genome integrity, their repetitive nature has thwarted dissection of local heterochromatin assembly and maintenance mechanisms. By engineering fission yeast strains carrying fluorescent reporters at a single subtelomere, we uncovered distinct subdomains. These subdomains have different silencing requirements: telomere-proximal regions rely on canonical shelterin- or RNAi-dependent nucleation pathways, whereas telomere-distal regions involve nucleosome remodelers, histone chaperones, and boundary-associated factors. Subdomains also exhibit discrete epigenetic states emerging both at homologous loci on different chromosome arms and along the same subtelomeric sequence. We document these epigenetic states using multi-generational live imaging and targeted perturbations. These analyses show that subtelomeric subdomains display position-specific, clonally variable silencing across a spectrum from robust to fragile epigenetic states. Interestingly, structural variants, common in subtelomeric sequences across eukaryotes, dictate local epigenetic stability. These findings reveal that subtelomeres, long recognized for their sequence variability, form a dynamic mosaic of coexisting epigenetic domains. This implies a wide range of gene-repressive regulatory logic at the chromosome ends, from environmental responsiveness to silencing stability.

CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo–electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity.

DNase I is a nonspecific endonuclease that preferentially cleaves double-stranded DNA (dsDNA) over single-stranded DNA (ssDNA) in the presence of Ca²⁺ and Mg²⁺. Although the structure and biochemical properties of DNase I are well-characterized, the catalytic process remains poorly understood, as earlier studies primarily inferred cleavage from end point fragments or time-averaged measurements rather than direct visualization. Here, we employ high-speed atomic force microscopy (HS-AFM) to directly visualize DNase I activity on linear dsDNA, circular dsDNA plasmids, and circular ssDNA plasmids. DNase I, observed as monomeric particles, dimers, and trimeric or higher-order aggregates, dynamically binds to and slides along DNA substrates while inducing both single-strand and double-strand cleavage events. These observations reveal that DNase I-mediated cleavage is not restricted to an isolated monomeric state and that sliding-like DNA-bound motion can accompany nonspecific nuclease activity. DNase I exhibits significantly higher cleavage efficiency toward dsDNA than ssDNA. Together, these results provide direct mechanistic insights into DNase I-mediated nucleic acid degradation, with implications for its biochemical functions and therapeutic applications.

Prokaryotic organisms have evolved unique strategies to acquire immunity against the constant threat of bacteriophage (phage) and mobile genetic elements. Hna is a broadly distributed anti-phage immune system that confers resistance against diverse phage by eliciting an abortive infection response. Using a combination of biochemistry, cryo-electron microscopy, and single-molecule fluorescence imaging, we reveal that Hna functions as a 3’—5’ single-stranded DNA exonuclease that forms an auto-inhibited dimer under physiological ATP concentrations. Biochemical and mutational analyses demonstrate that Hna catalytic outputs are governed by kinetic partitioning between ATPase and nuclease active sites. Disruption of this balance enhances DNA cleavage and causes cellular toxicity. Furthermore, we show that a phage-encoded single-stranded DNA-binding protein (5 A SSB) destabilizes the autoinhibited Hna dimer and shifts catalytic partitioning toward dysregulated nuclease activation. Conversely, phage escape mutants encode SSB variants that evade Hna surveillance by adopting higher order stoichiometries with enhanced DNA binding affinity. Our work establishes the molecular basis of Hna-mediated anti-phage activity and provides insights into how phage-encoded proteins can directly stimulate a bacterial immune response.

CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid preamplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas trans-nuclease detection of amplified products), a method that integrates isothermal linear DNA amplification with Cas12a detection in a single reaction. CATNAP uses a nicking enzyme and DNA polymerase to continuously generate single-stranded DNA, activating Cas12a's trans-cleavage activity without damaging the template. We optimized enzyme combinations, buffer conditions, and target selection to achieve high catalytic efficiency. CATNAP successfully distinguished between high- and low-risk HPV strains and detects HPV-16 in crude cell lysates of cervical cancer cells with minimal equipment, offering advantages over PCR-based approaches. We conclude that CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity, making accurate disease detection more accessible in resource-limited settings.

Prokaryotic organisms have evolved unique strategies to acquire immunity against the constant threat of bacteriophage (phage) and mobile genetic elements. Hna is a broadly distributed anti-phage immune system that confers resistance against diverse phage by eliciting an abortive infection response. Using a combination of biochemistry, cryo-electron microscopy, and single-molecule fluorescence imaging, we reveal that Hna functions as a 3’—5’ single-stranded DNA exonuclease that forms an auto-inhibited dimer under physiological ATP concentrations. We observed that Hna autoinhibition can be overcome by incorporation of a phage-encoded single-stranded DNA binding protein (SSB), stimulating unregulated Hna nuclease activity. Furthermore, phage escape mutants encode SSB variants that evade Hna surveillance by adopting higher order stoichiometries with enhanced DNA binding affinity. Our work establishes the molecular basis of Hna-mediated anti-phage activity and provides novel insights into how phage-encoded proteins can directly stimulate a bacterial immune response.

Retrons can produce multicopy single-stranded DNA in cells through self-primed reverse transcription. However, their potential for inserting genetic cargos in eukaryotes remains largely unexplored. Here we report the discovery and engineering of highly efficient retron-based gene editors for mammalian cells and vertebrates. Through bioinformatic analysis of metagenomic data and functional screening, we identify retron reverse transcriptases that are highly active in mammalian cells. Rational design further improves the editing efficiency to levels comparable with conventional single-stranded oligodeoxynucleotide donors but from a genetically encoded cassette. Retron editors exhibit robust activity with Cas12a nuclease and Cas9 nickase, expanding the genomic target scope and bypassing the need for a DNA double-stranded break. Using a rationally engineered retron editor, we incorporate a split GFP epitope tag for live-cell imaging. Lastly, we develop an all-RNA delivery strategy to enable DNA-free gene editing in cells and vertebrate embryos. This work establishes retron editors as a versatile and efficient tool for precise gene editing.

Subtelomeres are imperfect repeats adjacent to telomeres that are repressed by heterochromatin. Although essential for genome integrity, their repetitive nature has thwarted dissection of local heterochromatin assembly and maintenance mechanisms. Here, we engineered Schizosaccharomyces pombe strains carrying fluorescent reporters at a single subtelomere. We find that subtelomeric heterochromatin is organized into discrete subdomains that nucleate at telomere-proximal and cryptic internal sites. Telomere-proximal regions depend on canonical shelterin or RNA interference nucleation pathways, while telomere-distal regions require nucleosome remodelers, histone chaperones, and boundary-associated factors. Using multi-generational live imaging and targeted perturbations, we show that subtelomeric subdomains display position-specific, clonally variable silencing across a spectrum of robust to fragile epigenetic states. This clonal variegation is also induced by naturally occurring subtelomeric structural variants. These findings demonstrate that subtelomeric heterochromatin maintenance is not uniform but rather governed by local chromatin context and architecture.

CRISPR-Cas13d is increasingly used for RNA knockdowns due to its programmability, but off-target RNA binding and cleavage of near-cognate RNAs hinder its broader adoption. Here, we explore the mechanisms of nuclease activation by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal a series of active, intermediate, and inactive states that illustrate a detailed activation mechanism. The crRNA undergoes dramatic conformational changes upon target RNA binding, with the helical-1 domain transitioning from an initially docked state with the N-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics reveal that a single proximal mismatch preserves nanomolar binding affinity but completely abolishes nuclease activity by trapping Cas13d in an inactive state. We identify an active site loop in the HEPN domains that regulates substrate accessibility, with alanine scanning mutagenesis revealing both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d’s exquisite mismatch surveillance and provide a mechanistic framework for engineering RNA-targeting specificity and activity across HEPN nuclease family members.

CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid diagnostics enable rapid, sensitive pathogen detection. Cas12a is frequently used in these assays because target-activated trans cleavage of a reporter molecule generates an easily detectable signal. However, variable activity across assays suggests that the catalytic potential of Cas12a has been limited via unknown mechanisms. Here, we show that Cas12a trans-nuclease activity is auto-inhibited by long PAM-proximal DNA (>120 bp) following cis-cleavage of targets. Short targets (<100 bp), optimized trans cleavage substrates, and low salt buffers unleash high catalytic efficiency (≈108 M−1 s−1) and turnover (≈1 s−1) across Cas12a orthologs. Pooling multiple Cas12a ribonucleoproteins (RNPs) targeting clustered protospacers overcomes cis-cleavage auto-inhibition, further boosting sensitivity. Optimized CRISPR RNA pools enable sub-femtomolar sensitivity for target detection without any pre-amplification. This mechanistic insight and mitigation strategy broaden the application of CRISPR–Cas enzymes for nucleic acid diagnostics.