Processing and Visualization of Protec-Seq Data for the Generation of Calibrated, Genome-Wide Double Double-Strand Break (dDSB) Maps.

This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.

Harnessing noncanonical trans-cleavage characteristics of Cas12 and Cas13a to enhance CRISPR-based diagnostics.

Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans-cleavage activities, yet their activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, and Cas13a, uncovering the characteristics of their trans-DNase and trans-RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target for CRISPR-Cas13a, markedly increasing the detection sensitivity for single-base mismatches. Moreover, the trans-cleavage activities of Cas12a and Cas13a can be activated by diverse RNA:DNA and RNA:RNA duplexes, respectively, indicating that the presence of stem-loop structures in crRNAs is not essential for their activation. Notably, Cas12f1, unlike Cas12a, exhibits intrinsic RNase activity independently of activation. Leveraging these insights, we have improved the accuracy of a dual-gene target detection approach that employs the CRISPR-Cas12f1 and Cas13a systems. Our research advances the understanding of the noncanonical activation characteristics of Cas12 and Cas13a, contributing to the field of CRISPR-based diagnostics.

Endonuclease G is dispensable for sperm mitochondrial DNA elimination during spermatogenesis in mice.

Maternal inheritance of mitochondrial DNA (mtDNA) is a widespread phenomenon in eukaryotes. Our earlier research indicated that sperm mtDNA is removed prior to fertilization in mice, and Endonuclease G (ENDOG) orchestrates the degradation of sperm mitochondria in Caenorhabditis elegans. However, the mechanisms underlying sperm mtDNA disposal in mammals remain poorly understood. To investigate the potential role of ENDOG in sperm mtDNA elimination, we created Endog knockout (Endog-/-) mice. Our findings revealed that Endog-/- mice maintained normal spermatogenesis and fertility. Most strikingly, we detected no substantial discrepancy in sperm mtDNA copy number between Endog-/- and control mice. Furthermore, we noted that sperm mtDNA copy numbers were unchanged in both less motile and motile sperm isolated by Percoll gradient centrifugation from Endog-/- and control mice. Taken together, our results indicate that ENDOG is not essential for spermatogenesis or the elimination of sperm mtDNA in mice.

Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system.

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells.

Expression and purification of cell-penetrating Cas9 and Cas12a enzymes for peptide-assisted genome editing.

Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells.

Enhanced CRISPR/Cas12a Fluorimetry via a DNAzyme-Embedded Framework Nucleic Acid Substrate.

CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.

High-contrast imaging of cellular non-repetitive drug-resistant genes via in situ dead Cas12a-labeled PCR.

In situ imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence in situ hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes. We employed a syncretic dCas12a-green fluorescent protein (dCas12a-GFP) to tag the amplicons, thereby enabling high-contrast imaging and avoiding multiple fluorescently labeled probes. The CasPCR assay can quantify quinolone-resistant Salmonella enterica in mixed populations and identify them isolated from poultry farms.

Exploring the removal of Spo11 and topoisomerases from DNA breaks in S. cerevisiae by human Tyrosyl DNA Phosphodiesterase 2.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos-the products of nucleolytic removal by Mre11-have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2-a protein known to repair DNA ends arising from abortive topoisomerase activity-on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo-contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination.

Exploring RNA-guided DNA scissors in eukaryotes: Are Fanzors counterparts of CRISPR-Cas12s?

Fanzors are recently characterized RNA-guided DNA endonucleases found in eukaryotic organisms. In this issue of Cell, Xu, Saito et al. reveal the structural diversity of Fanzors and identify key features shared with TnpB and Cas12 proteins, providing a comprehensive perspective on their molecular function and evolution.

DNase II Can Efficiently Digest RNA and Needs to Be Redefined as a Nuclease.

DNase II, identified in 1947 and named in 1953, is an acidic DNA endonuclease prevalent across organisms and crucial for normal growth. Despite its expression in nearly all human tissues, as well as its biological significance, DNase II's detailed functions and corresponding mechanisms remain unclear. Although many groups are trying to figure this out, progress is very limited. It is very hard to connect its indispensability with its DNA cleavage activity. In this study, we find that DNase II secreted to saliva can digest RNA in mildly acidic conditions, prompting us to hypothesize that salivary DNase II might digest RNA in the stomach. This finding is consistent with the interesting discovery reported in 1964 that RNA could inhibit DNase II's activity, which has been largely overlooked. This RNA digestion activity is further confirmed by using purified DNase II, showing activity to digest both DNA and RNA effectively. Here, we suggest redesignating DNase II as DNase II (RNase). The biological functions of DNase II are suggested to recycle intracellular RNA or digest external nucleic acids (both RNA and DNA) as nutrients. This discovery may untangle the mystery of DNase II and its significant biofunctions.

Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.

Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68, the Drosophila homolog of Spo11, to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region.

Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA.

The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system's specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings.

Synergistic effect of split DNA activators of Cas12a with exon-unwinding and induced targeting effect.

CRISPR-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a-RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.

Apoptotic endonuclease EndoG induces alternative splicing of Caspase-2.

Caspase-2 (Casp-2) is an enzyme that regulates the development of apoptosis upon alternative splicing of its mRNA. The long form of Casp-2 (Casp-2L) promotes apoptosis while the short form (Casp-2S) has decreased enzymatic activity and inhibits the development of apoptotic processes. However, very little is known about the mechanism of Casp-2 alternative splicing. Several endonucleases are known to participate in this process. The aim of this study was to determine the role of EndoG in regulation of Casp-2 alternative splicing. Strong correlation between expression levels of EndoG and Casp-2 splice-variants was found in CD4⁺ and CD8⁺ human T lymphocytes. Such correlation increased after incubation of these cells with etoposide. Increased expression of Casp-2S was determined during EndoG over-expression in CD4⁺ T-cells, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. Casp-2 alternative splicing was induced by a 60-mer RNA oligonucleotide in naked nuclei and in cells after transfection. The identified long non-coding RNA of 1016 nucleotides is the precursor of the 60-mer RNA oligonucleotide. Based on the results the following mechanism has been proposed. Casp-2 pre-mRNA is transcribed from the coding DNA strand while long non-coding RNA is transcribed from the template strand of the Casp-2 gene. EndoG digests long non-coding RNA and produces the 60-mer RNA oligonucleotide complementary to the Casp-2 pre-mRNA exon 9 and intron 9 junction place. Interaction of the 60-mer RNA oligonucleotide and Casp-2 pre-mRNA causes alternative splicing.

Yeast EndoG prevents genome instability by degrading extranuclear DNA species.

In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.

Structural basis for DNA recognition by a viral genome-packaging machine.

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii.

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within  30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

Neuronal double-stranded DNA accumulation induced by DNase II deficiency drives tau phosphorylation and neurodegeneration.

BACKGROUND: Deoxyribonuclease 2 (DNase II) plays a key role in clearing cytoplasmic double-stranded DNA (dsDNA). Deficiency of DNase II leads to DNA accumulation in the cytoplasm. Persistent dsDNA in neurons is an early pathological hallmark of senescence and neurodegenerative diseases including Alzheimer's disease (AD). However, it is not clear how DNase II and neuronal cytoplasmic dsDNA influence neuropathogenesis. Tau hyperphosphorylation is a key factor for the pathogenesis of AD. The effect of DNase II and neuronal cytoplasmic dsDNA on neuronal tau hyperphosphorylation remains unclarified. METHODS: The levels of neuronal DNase II and dsDNA in WT and Tau-P301S mice of different ages were measured by immunohistochemistry and immunolabeling, and the levels of DNase II in the plasma of AD patients were measured by ELISA. To investigate the impact of DNase II on tauopathy, the levels of phosphorylated tau, phosphokinase, phosphatase, synaptic proteins, gliosis and proinflammatory cytokines in the brains of neuronal DNase II-deficient WT mice, neuronal DNase II-deficient Tau-P301S mice and neuronal DNase II-overexpressing Tau-P301S mice were evaluated by immunolabeling, immunoblotting or ELISA. Cognitive performance was determined using the Morris water maze test, Y-maze test, novel object recognition test and open field test. RESULTS: The levels of DNase II were significantly decreased in the brains and the plasma of AD patients. DNase II also decreased age-dependently in the neurons of WT and Tau-P301S mice, along with increased dsDNA accumulation in the cytoplasm. The DNA accumulation induced by neuronal DNase II deficiency drove tau phosphorylation by upregulating cyclin-dependent-like kinase-5 (CDK5) and calcium/calmodulin activated protein kinase II (CaMKII) and downregulating phosphatase protein phosphatase 2A (PP2A). Moreover, DNase II knockdown induced and significantly exacerbated neuron loss, neuroinflammation and cognitive deficits in WT and Tau-P301S mice, respectively, while overexpression of neuronal DNase II exhibited therapeutic benefits. CONCLUSIONS: DNase II deficiency and cytoplasmic dsDNA accumulation can initiate tau phosphorylation, suggesting DNase II as a potential therapeutic target for tau-associated disorders.

Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.

Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.

FIGNL1-FIRRM is essential for meiotic recombination and prevents DNA damage-independent RAD51 and DMC1 loading.

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.