Endonuclease Activity Research Articles

A split single-stranded DNA activator-based Cas12a fluorescence biosensor for specific H1N1 detection

We developed a novel CRISPR/Cas12a fluorescence biosensor to specifically detect the H1N1 virus based on splitting single-stranded DNA (ssDNA) activators. Specifically, activators 1 and 2 were generated from dual target nucleic acid sites to induce a three-way junction (3WJ) isothermal amplification process. Two specific H1N1 RNA nucleic acid sites were bound to a set of probes to form a 3WJ, which facilitated repeated extension and nicking reactions to produce high levels of split ssDNA activators via DNA polymerase and nicking endonuclease activity. Amplified activators 1 and 2 fully assembled with Cas12a/crRNA to form complexes. Activated Cas12a trans-cleaved ssDNA reporter molecules to generate fluorescence signals. Under optimal conditions, biosensor dynamic detection range was 1 pM–10 nM, and the detection limit was 205 fM. Thus, our biosensor showed high specificity, reliability, and robustness, which highlights its potential use for H1N1 diagnostics.

CRISPR/Cas9 Mechanism and Applications Progression

CRISPR (short for Clustered Regularly Interspaced Short Palindromic Repeats) and the accompanying endonuclease Cas9 are potent tools for gene editing in many applied disciplines. The CRISPR/Cas9 system primarily functions through two components, in which the single guide RNA (sgRNA) directs the Cas9 protein with endonuclease activity to carry out precise DNA cleavage. The cell's inherent repair mechanisms allow it to complete the subsequent reconnection of the broken genes. Gene knock-in or knock-out can therefore be achieved via this approach. This system was typically applied to prokaryotes in early studies. Its role as a component of their defense mechanism has been thoroughly studied. With rapid development over the past ten years, this system can now be effectively used in a multitude of eukaryotic cells, including yeast cells and mammalian cells. Because of its accuracy, effectiveness, and low cost, CRISPR/Cas9 has been widely used for human disease treatment and has shown great potential in gene therapies for hereditary diseases. However, since more late-stage clinical trials are required to validate its safety, its applications still face many challenges. This review investigates the mechanism of CRISPR/Cas9-mediated gene editing technology and highlights its recent applications in Alzheimer's disease, Sickle cell disease, and Duchenne muscular dystrophy, which are difficult to treat with current approaches. Some challenges the CRISPR/Cas9 system has encountered at this stage are described, and its prospects are also examined.

Detection of Single Nucleotide Polymorphisms of Circulating Tumor DNA by Strand Displacement Amplification Coupled with Liquid Chromatography.

The detection of multiple single nucleotide polymorphisms (SNPs) of circulating tumor DNA (ctDNA) is still a great challenge. In this study, we designed enzyme-assisted nucleic acid strand displacement amplification combined with high-performance liquid chromatography (HPLC) for the simultaneous detection of three ctDNA SNPs. First, the trace ctDNA could be hybridized to the specially designed template strand, which initiated the strand displacement nucleic acid amplification process under the synergistic action of DNA polymerase and restriction endonuclease. Then, the targets would be replaced with G-quadruplex fluorescent probes with different tail lengths. Finally, the HPLC-fluorescence assay enabled the separation and quantification of multiple signals. Notably, this method can simultaneously detect both the wild type (WT) and mutant type (MT) of multiple ctDNA SNPs. Within a linear range of 0.1 fM-0.1 nM, the detection limits of BRAF V600E-WT, EGFR T790M-WT, and KRAS 134A-WT and BRAF V600E-MT, EGFR T790M-MT, and KRAS 134A-MT were 29, 31, and 11 aM and 22, 29, and 33 aM, respectively. By using this method, the mutation rates of multiple ctDNA SNPs in blood samples from patients with lung or breast cancer can be obtained in a simple way, providing a convenient and highly sensitive analytical assay for the early screening and monitoring of lung cancer.

Abstract 3335: A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers

Abstract Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network called the unfolded protein response (UPR). Inositol-requiring enzyme 1α (IRE1α) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved way of the UPR. The IRE1α-XBP1 pathway, one of the most essential branches of the UPR, is activated in various types of cancer. When IRE1α is activated, the spliced form of XBP1 (XBP1s) is generated to activate the transcription of many pro-survival genes to prevent cellular death and relieve cellular stress. IRE1α plays an instrumental pro-tumoral role in various cancers, such as glioblastoma, myeloma, lung adenocarcinoma, and breast cancers, as well as high IRE1α activity is associated with poor prognoses. Moreover, IRE1-XBP1 plays an essential role in the UPR and ER stress responses, which have been implicated in the development of drug resistance. Thereby, blockade of the IRE1α-XBP1 pathway is crucial as it is considered as a potential anti-cancer strategy. Here, we developed a potent and selective IRE1α RNase inhibitor, which have a hydroxy-aryl-aldehydes (HAA) moiety that binds to IRE1α within the RNase catalytic site. Our lead compound, HM100168, potently inhibited the RNase activity of human IRE1α at nanomolar concentrations without affecting its kinase activity. Experiments were performed on MCF7 breast cancer cells to confirm the inhibitory effect in the mRNA and protein levels of XBP1s, which was increased by tunicamycin (TM), a ER stress inducer. HM100168 strongly inhibited ​​XBP1s levels in a dose-dependent manner, displaying the potent IC50 values. We tested cell growth inhibition activity in 3D as well as 2D cell culture condition of various cancer cell lines. Interestingly, HM100168 effectively inhibited cell proliferation of diverse cancer cell lines in both 2D and 3D culture condition. In addition, we explored the promising anti-cancer effects of HM100168 and its synergistic potential when combined with anti-tumorigenic agents. As a result, HM100168 in combination with anti-tumorigenic agents significantly enhanced suppression of tumor growth in human cancer cell transplanted mice. In conclusion, we have identified a novel compound with unique properties that specifically blocked the endonuclease activity of IRE1α. HM100168 inhibited IRE1α endonuclease activity, without affecting its kinase activity after endoplasmic reticulum stress in vitro. The identification of this novel IRE1α inhibitor supports the hypothesis that the IRE1α-XBP1 axis is a promising target for anticancer therapy. Also, co-targeting of IRE1α-XBP1 in combination with anti-tumorigenic agents can be considered a novel approach to overcome cancer resistance. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Citation Format: Jisook Kim, Seung Hyun Jung, Minjeong Kim, Wongi Park, Jooyun Byun, Soonki Park, Miyoung Lee, Yu-Yon Kim, Junghwa Park, Seokhyun Hong, Minhwa Kim, Young Gil Ahn. A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3335.

Abstract 1699: Defining the impact of aberrant transcription on the chromatin structure

Abstract Genome organization in humans is governed by two major mechanisms: loop extrusion by cohesin and CTCF and the spatial compartmentalization of the chromosomes. These folding mechanisms have been shown to regulate several genomic functions, including gene expression. However, how transcription might regulate the chromatin structure remains a subject of many ongoing investigations. In this study, we use the Integrator protein complex as a tool to understand the impact of aberrant transcription on the 3D structure of the genome. During transcription initiation, RNA Polymerase-II (Pol-II) often pauses proximally to the promoter before further elongating transcripts. The Integrator protein complex regulates this checkpoint by its endonuclease and phosphatase activity. This protein complex is essential in premature transcription termination in hundreds of protein-coding genes. It also facilitates the synthesis of non-coding (ncRNA), such as enhancer RNAs (eRNAs) and long non-coding RNAs (lncRNAs), which have been shown to support loop extrusion domains, and their absence may disrupt these domains. Using HiC sequencing, we found that depleting Integrator proteins can interfere with forming and maintaining loop domains. This study aims to be the first to decipher the relationship between chromosome structure and Integrator protein activity and address our very little knowledge of how misregulation of transcription termination may influence the folding of chromosomes. Citation Format: Naiju Thomas, Timothy E. Reznicek, Erez Lieberman Aiden, M. Jordan Rowley, Eric Wagner, Guy Nir. Defining the impact of aberrant transcription on the chromatin structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1699.

Abstract 3016: MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development

Abstract Introduction: MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein), also known as Regnase-1, belongs to the CCCH zinc finger protein family, featuring a PilT N-terminus (PIN) domain-like RNase domain. Utilizing its intrinsic endonuclease activity, MCPIP1 functions as a precise modulator of cytokines, exhibiting an anti-inflammatory effect through the targeted degradation of specific mRNA transcripts. While recent studies have highlighted MCPIP1 as a potential tumor suppressor with decreased levels in breast cancer and clear cell renal cell carcinoma, its role in hepatocellular carcinoma (HCC) development remains unexplored. Material and Methods: To investigate the role of MCPIP1 in HCC development, we used tissue-specific knockout mice with the deletion of Mcpip1 in hepatocytes. HCC was induced by administering diethylnitrosamine (DEN) to two-week-old mice. We conducted a comprehensive analysis, including the assessment of EMT and fibrosis markers, proinflammatory cytokines, and immune cell infiltration, using Western blot (WB), mass spectrometry, quantitative PCR (qPCR), and multiple immunohistochemistry (IHC) stainings. To identify differences between the wild-type (wt) and Mcpip1 knockout (KO) mice groups more effectively, hepatocytes were isolated using the perfusion method, and RNA-Seq analysis was performed. Results and Discussion: The tissue-specific knockout of Mcpip1 in the liver has notable molecular consequences. Transcriptomic profiling revealed an upregulation of transcripts integral to tumor development, including Cxcr4, Met, Cxcl12, Hgf, and Src, alongside factors characteristically associated with cancer stem cells, Myc, Klf4, and CD133. The absence of MCPIP1 triggered immune cell activation in the liver, as evidenced by an augmented expression of markers specific to myeloid and lymphoid cell populations, indicative of an intensified immunological response. IHC examination of liver tissue from Mcpip1-deficient mice revealed significant fibrotic alterations, characterized by increased collagen deposition, and enhanced expression of alpha-smooth muscle actin (αSMA). The lack of Mcpip1 influences the increased levels of vimentin, fibronectin, and connective tissue growth factor (cTGF), indicating enhanced tissue remodeling. Conclusions: MCPIP1, recognized for its anti-inflammatory functions, appears to function as a potential tumor suppressor in HCC development. The lack of MCPIP1 results in the upregulation of transcripts linked to tumorigenesis, immune cell activation, and notable fibrotic changes, highlighting its involvement in the complex molecular dynamics of HCC progression. This study was supported by National Science Center grants no. 2017/26/E/NZ5/00691, 2021/41/N/NZ4/04187. Citation Format: Katarzyna Miekus, Oliwia Kwapisz, Paulina Marona, Judyta Gorka, Jerzy Kotlinowski, Ewelina Pospiech, Jolanta Jura. MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3016.

Structure-specific DNA endonuclease T7 endonuclease I cleaves DNA containing UV-induced DNA lesions.

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs, and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication, and repair in E.coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analyzed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD), and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7 endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.

Label-free colorimetric detection platform based on catalytic hairpin self-assembly and G-quadruplex/hemin DNAzyme for comprehensive biomarker profiling

The expression level of human apurinic/apyrimidinic endonuclease 1 (APE1) is closely associated with the onset of various diseases, establishing it as a crucial clinical biomarker and a target in anti-cancer efforts. This study accomplished colorimetric and visual detection of APE1 by harnessing its endonuclease activity through catalytic hairpin self-assembly (CHA) and G-quadruplex/hemin DNAzyme. Optimization of the freedom degrees of the G-rich sequence significantly improved the detection performance of the strategy by influencing DNAzyme formation. Additionally, we replaced the signal reporting system with a molecular beacon to develop a fluorescence detection strategy, which served as an extension of the signal amplification system for validation and signal readout. The fluorescent probe method achieved a detection limit of 3.37 × 10−4 U/mL, while the colorimetric method yielded a detection limit of 6.5 × 10−3 U/mL, with a linear range spanning from 0.01 to 0.25 U/mL. Subsequently, the colorimetric approach effectively assessed APE1 activity in biological samples and facilitated the screening of APE1 activity inhibitors. Furthermore, this CHA/G-quadruplex/hemin DNAzyme strategy was adapted for the colorimetric detection of adenosine, showcasing its broad applicability across various biomarkers. The developed colorimetric analytical strategy represents a pivotal biosensing platform for diagnosing and treating diseases.

Investigating the Merits of Microfluidic Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS) in the Bottom-Up Characterization of Larger RNAs.

Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on the strand-cleavage activity of nucleotide-specific endonucleases to generate smaller oligonucleotides amenable to gas-phase sequencing. The complexity of these hydrolytic mixtures calls for the utilization of a front-end separation to facilitate full mass spectrometric (MS) characterization. This report explored the merits of microfluidic capillary zone electrophoresis (CZE) as a possible alternative to common liquid chromatography techniques. An oligonucleotide ladder was initially employed to investigate the roles of fundamental analyte features and experimental parameters in determining the outcome of CZE-MS analyses. The results demonstrated the ability to fully resolve the various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that was visibly affected by the overall amount of material injected into the system. Analogous results were obtained from a digestion mixture prepared by treating yeast tRNAPhe (75 nt) with RNase T1, which provided several well-resolved peaks in spite of the increasing sample heterogeneity. The regular shapes of such peaks, however, belied the fact that most of them contained sets of comigrating species, as shown by the corresponding MS spectra. Even though it was not possible to segregate each species into an individual electrophoretic peak, the analysis still proved capable of unambiguously identifying a total of 29 hydrolytic products, which were sufficient to cover 96% of the tRNAPhe's sequence. Their masses accurately reflected the presence of modified nucleotides characteristic of this type of substrate. The analysis of a digestion mixture obtained from the 364 nt HIV-1 5'-UTR proved to be more challenging. The electropherogram displayed fewer well-resolved peaks and significantly greater incidence of product comigration. In this case, fractionating the highly heterogeneous mixture into discrete bands helped reduce signal suppression and detection bias. As a result, the corresponding MS data enabled the assignment of 248 products out of the possible 513 predicted from the 5'-UTR sequence, which afforded 100% sequence coverage. This figure represented a significant improvement over the 36 total products identified earlier under suboptimal conditions, which afforded only 57% coverage, or the 83 observed by direct infusion nanospray-MS (72%). These results provided a measure of the excellent potential of the technique to support the bottom-up characterization of progressively larger NA samples, such as putative NA therapeutics and mRNA vaccines.

Biochemical characterization and mutational analysis of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Archaeal NurA protein plays a key role in producing 3′-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5′–3′ exonuclease activity for degrading DNA, displaying maximum efficiency at 45oC ∼ 65oC and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques.Key points• Non-switchable methylases always inhibit the relevant type IIS endonuclease activity• Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site• Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assemblyGraphical

Purification of Saccharomyces cerevisiae recombinant Crp1

A complex Mus81-Mm4 is a DNA structure–specific endonuclease in Saccharomyces cerevisiae. Mus81-Mms4 functions in processing of recombination intermediates that could arise during the repair of stalled and blocked replication forks and double stranded breaks. Mus81-Mms4 works with many proteins involved in DNA repair, replication fork stability, and joint molecule formation/resolution during homologous recombination repair. A biochemical screening of protein(s) that enhances the Mus81-Mms4 endonuclease activity on its preferable substrates in vitro revealed that Crp1, a cruciform DNA-recognizing protein, which can specifically bind to DNA four-way junction structures like Holliday junctions could be the potential factor. To further demonstrate that Crp1 interacts functionally with Mus81-Mms4 in vitro, we carried out the purification of recombinant Crp1 using Escherichia coli system. Our results showed that the purified Crp1 was highly homogenous and active that is ready for biochemical use.

Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

Optimization and biological evaluation of l-DOPA derivatives as potent influenza PAN endonuclease inhibitors with multi-site binding characteristics

Emerging and potential influenza pandemics still are an enormous worldwide public health challenge. The PAN endonuclease has been proved to be a promising target for anti-influenza drug design. Here, we report the discovery and optimization of potent Y-shaped PAN inhibitors featuring multi-site binding characteristics with l-DOPA as a starting point. We systematically modified the hit 1 bearing two-binding characteristics based on structure-based rational design combined with multisite binding and conformational constraint strategies, generating four families of l-DOPA derivatives for SARs analysis. Among these substances, N, 3-di-substituted 1, 2, 3, 4-tetrahydroisoquinoline derivative T-31 displayed superior properties as a lead PAN endonuclease inhibitor and antiviral agent. The lead T-31 inhibited PAN endonuclease activity with an IC50 value of 0.15 μM and showed broad and submicromolar anti-influenza potency in cell-based assays. More importantly, T-31 could simultaneously target both influenza HA and the RdRp complex, thus interfering with virus entry into host cells and viral replication. This study offers a set of novel PAN endonuclease inhibitors with multi-site binding characteristics starting from the l-DOPA skeleton.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

Evaluation of restriction and Cas endonuclease kinetics using matrix-insensitive magnetic biosensors

The enzymatic actions of endonucleases in vivo can be altered due to bound substrates and differences in local environments, including enzyme concentration, pH, salinity, ionic strength, and temperature. Thus, accurate estimation of enzymatic reactions in vivo using matrix-dependent methods in solution can be challenging. Here, we report a matrix-insensitive magnetic biosensing platform that enables the measurement of endonuclease activity under different conditions with varying pH, salinity, ionic strength, and temperature. Using biosensor arrays and orthogonal pairs of oligonucleotides, we quantitatively characterized the enzymatic activity of EcoRI under different buffer conditions and in the presence of inhibitors. To mimic a more physiological environment, we monitored the sequence-dependent star activity of EcoRI under unconventional conditions. Furthermore, enzymatic activity was measured in cell culture media, saliva, and serum. Last, we estimated the effective cleavage rates of Cas12a on anchored single-strand DNAs using this platform, which more closely resembles in vivo settings. This platform will facilitate precise characterization of restriction and Cas endonucleases under various conditions.

Intein-based thermoregulated meganucleases for containment of genetic material.

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18°C and not at higher temperatures. We constructed an orthogonal set of temperature-sensitive meganucleases (TSMs) by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18°C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. TSMs eliminated plasmids from E. coli Nissle 1917 after passage through the mouse gut when fecal resuspensions were incubated at 18°C but not at 37°C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

Formation and Properties of DNA Adducts Generated by Reactions of Abasic Sites with 1,2-Aminothiols Including Cysteamine, Cysteine Methyl Ester, and Peptides Containing N-Terminal Cysteine Residues.

The reaction of 1,2-aminothiol groups with aldehyde residues in aqueous solution generates thiazolidine products, and this process has been developed as a catalyst-free click reaction for bioconjugation. The work reported here characterized reactions of the biologically relevant 1,2-aminothiols including cysteamine, cysteine methyl ester, and peptides containing N-terminal cysteine residues with the aldehyde residue of apurinic/apyrimidinic (AP) sites in DNA oligomers. These 1,2-aminothiol-containing compounds rapidly generated adducts with AP sites in single-stranded and double-stranded DNA. NMR and MALDI-TOF-MS analyses provided evidence that the reaction generated a thiazolidine product. Conversion of an AP site to a thiazolidine-AP adduct protected against the rapid cleavage normally induced at AP sites by the endonuclease action of the enzyme APE1 and the AP-lyase activity of the biogenic amine spermine. In the presence of excess 1,2-aminothiols, the thiazolidine-AP adducts underwent slow strand cleavage via a β-lyase reaction that generated products with 1,2-aminothiol-modified sugar residues on the 3'-end of the strand break. In the absence of excess 1,2-aminothiols, the thiazolidine-AP adducts dissociated to release the parent AP-containing oligonucleotide. The properties of the thiazolidine-AP adducts described here mirror critical properties of SRAP proteins HMCES and YedK that capture AP sites in single-stranded regions of cellular DNA and protect them from cleavage.

Hsa_circ_0001583 fuels bladder cancer metastasis by promoting staphylococcal nuclease and tudor domain containing 1-mediated MicroRNA decay

Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa_circ_0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa_circ_0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa_circ_0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa_circ_0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa_circ_0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa_circ_0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa_circ_0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets.