Harnessing noncanonical crRNA for highly efficient genome editing.

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

The Terminase Complex of Each Human Herpesvirus.

The human herpesviruses (HHVs) are classified into the following three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. These HHVs have distinct pathological features, while containing a highly conserved viral replication pathway. Among HHVs, the basic viral particle structure and the sequential processes of viral replication are nearly identical. In particular, the capsid formation mechanism has been proposed to be highly similar among herpesviruses, because the viral capsid-organizing proteins are highly conserved at the structural and functional levels. Herpesviruses form capsids containing the viral genome in the nucleus of infected cells during the lytic phase, and release infectious virus (i.e., virions) to the cell exterior. In the capsid formation process, a single-unit-length viral genome is encapsidated into a preformed capsid. The single-unit-length viral genome is produced by cleavage from a viral genome precursor in which multiple unit-length viral genomes are tandemly linked. This encapsidation and cleavage is carried out by the terminase complex, which is composed of viral proteins. Since the terminase complex-mediated encapsidation and cleavage is a virus-specific mechanism that does not exist in humans, it may be an excellent inhibitory target for anti-viral drugs with high virus specificity. This review provides an overview of the functions of the terminase complexes of HHVs.

Double-negative B cells and DNASE1L3 colocalise with microbiota in gut-associated lymphoid tissue.

Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.

Phosphorothioate-modified G-quadruplex as a signal-on dual-mode reporter for CRISPR/Cas12a-based portable detection of environmental pollutants.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-powered biosensor with a G-quadruplex (G4) reporter offer the benefits of simplicity and sensitivity, making them extensively utilized in detection applications. However, these biosensors used for monitoring pollutants in environmental water samples may face the problem of high background signal and easy interference due to the "signal-off" output. It is obvious that a biosensor based on the CRISPR/Cas12a system and G4 with a "signal on" output mode needs to be designed for detecting environmental pollutants. RESULTS: By using phosphorothioate-modified G4 as a reporter and catalytic hairpin assembly (CHA) integrated with Cas12a as an amplification strategy, a "signal-on" colorimetric/photothermal biosensor (psG4-CHA/Cas) for portable detection of environmental pollutants was developed. With the help of functional nucleotides, the target pollutant (kanamycin or Pb2+) triggers a CHA reaction to produce numerous double-strand DNA, which can activate Cas12a's trans-cleavage activity. The active Cas12a cleaves locked DNA to release caged psG-rich sequences. Upon binding hemin, the psG-rich sequence forms a psG4/hemin complex, facilitating the oxidation of the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into the blue photothermal agent (oxTMB). The smartphone was employed for portable colorimetric detection of kanamycin and Pb2+. The detection limits were found to be 100 pM for kanamycin and 50 pM for Pb2+. Detection of kanamycin and Pb2+ was also carried out using a portable thermometer with a detection limit of 10 pM for kanamycin and 8 pM for Pb2+. SIGNIFICANCE: Sensitive, selective, simple and robust detection of kanamycin and Pb2+ in environmental water samples is achieved with the psG4-CHA/Cas system. This system not only provides a new perspective on the development of efficient CRISPR/Cas12a-based "signal-on" designs, but also has a promising application for safeguarding human health and environmental monitoring.

Pirh2 modulates the mitochondrial function and cytochrome c-mediated neuronal death during Alzheimer's disease.

Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key pathological contributor to Alzheimer's disease (AD), therefore, the role of Pirh2 was investigated in streptozotocin and oligomer Aß1-42 induced rodent experimental model of AD. Pirh2 protein abundance increased during AD conditions, and transient silencing of Pirh2 inhibited the disease-specific pathological markers like level of p-Tau, ßamyloid, acetylcholinesterase activity, and neuronal death. Biochemically, Pirh2 silencing significantly attenuated the oxidative stress, depleted mitochondrial membrane potential, cytochrome c translocation from mitochondria to cytosol, and depleted mitochondrial complex-I activity, and ATP level. Pirh2 silencing also inhibited the altered level of VDAC1, hsp75, hexokinase1, t-Bid, caspase-9, and altered level of apoptotic proteins (Bcl-2, Bax). MALDI-TOF/TOF, co-immunoprecipitation, and UbcH13-linked ubiquitylation assay confirmed the interaction of Pirh2 with cytochrome c and the role of Pirh2 in ubiquitylation of cytochrome c, along with Pirh2-dependent altered proteasome activity. Additionally, Pirh2 silencing further inhibited the translocation of mitochondrion-specific endonuclease G and apoptosis-inducing factors to the nucleus and DNA damage. In conclusion, findings suggested the significant implication of Pirh2 in disease pathogenesis, particularly through impaired mitochondrial function, including biochemical alterations, translocation of cytochrome c, endonuclease G and apoptosis-inducing factor, DNA damage, and neuronal apoptosis.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA.

BACKGROUND: CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. RESULTS: Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. SIGNIFICANCE: The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

Low-cost inkjet-printed nanostructured biosensor based on CRISPR/Cas12a system for pathogen detection.

The escalating global incidence of infectious diseases caused by pathogenic bacteria, especially in developing countries, emphasises the urgent need for rapid and portable pathogen detection devices. This study introduces a sensitive and specific electrochemical biosensing platform utilising cost-effective electrodes fabricated by inkjet-printing gold and silver nanoparticles on a plastic substrate. The biosensor exploits the CRISPR/Cas12a system for detecting a specific DNA sequence selected from the genome of the target pathogen. Upon detection, the trans-activity of Cas12a/gRNA is triggered, leading to the cleavage of rationally designed single-strand DNA reporters (linear and hairpin) labelled with methylene blue (ssDNA-MB) and bound to the electrode surface. In principle, this sensing mechanism can be adapted to any bacterium by choosing a proper guide RNA to target a specific sequence of its DNA. The biosensor's performance was assessed for two representative pathogens (a Gram-negative, Escherichia coli, and a Gram-positive, Staphylococcus aureus), and results obtained with inkjet-printed gold electrodes were compared with those obtained by commercial screen-printed gold electrodes. Our results show that the use of inkjet-printed nanostructured gold electrodes, which provide a large surface area, in combination with the use of hairpin reporters containing a poly-T loop can increase the sensitivity of the assay corresponding to a signal variation of 86%. DNA targets amplified from various clinically isolated bacteria, have been tested and demonstrate the potential of the proposed platform for point-of-need applications.

Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.

Three-dimensional DNA nanomachine biosensor coupled with CRISPR Cas12a cascade amplification for ultrasensitive detection of carcinoembryonic antigen.

The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited accessibility and specificity. This study proposes a rapid and convenient Cas12a-based assay for the direct detection of CEA in clinical serum samples, aiming to address these limitations. The protocol involves a rolling machine operation, followed by a 5-min Cas12a-mediated cleavage process. The assay demonstrates the capability to detect human serum with high anti-interference performance and a detection limit as low as 0.2 ng/mL. The entire testing procedure can be accomplished in 75 min without centrifugation steps, and successfully reduced the limit of detection of traditional DNA walking machine by 50 folds. Overall, the testing procedure can be easily implemented in clinical settings.

The deficiency of DNASE1L3 does not affect systemic sclerosis pathogenesis in two inducible murine models of the disease.

We induced systemic sclerosis (SSc)-like disease in both wild-type and Dnase1l3-deficient mice using two distinct approaches involving bleomycin and hypochlorous acid injections. Our observations revealed that the deficiency in DNASE1L3 did not affect tissue fibrosis or inflammation caused by these treatments. Despite the association of single nucleotide polymorphisms in humans with SSc pathogenesis, our study demonstrates that DNASE1L3 is dispensable in two inducible murine models of SSc-like pathogenesis.

Posttranscriptional regulation of FAN1 by miR-124-3p at rs3512 underlies onset-delaying genetic modification in Huntington's disease.

Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-ß-lactamase (NDM) are particularly concerning due to their resistance to most ß-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention. METHODS: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples). RESULTS: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 100 copies/µL for fluorescence assay and 101 copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements. CONCLUSIONS: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

Prophage terminase with tRNase activity sensitizes Salmonella enterica to oxidative stress.

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNALeu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Label/immobilization-free Cas12a-based electrochemiluminescence biosensor for sensitive DNA detection.

Electrochemiluminescence (ECL) is one of the most sensitive techniques in the field of diagnostics. However, they typically require luminescent labeling and electrode surface biological modification, which is a time-consuming and laborious process involving multiple steps and may also lead to low reaction efficiency. Fabricating label/modification-free biosensors has become one of the most attractive parts for simplifying the ECL assays. In this work, the ECL luminophores carbon dots (CDs) were encapsulated in DNA hydrogel in situ by a simple rolling circle amplification (RCA) reaction. Upon binding of the target DNA, active Cas12a induces a collateral cleavage of the hydrogel's ssDNA backbone, resulting in a programmable degradation of the hydrogel and the release of CDs. By directly measuring the released CDs ECL, a simple and rapid label/modification-free detection of the target HPV-16 was realized. It is noted that this method allowed for 0.63 pM HPV-16 DNA detection without any amplification step, and it could take only ∼60 min for a fast test of a human serum sample. These results showed that our label/modification-free ECL biosensor has great potential for use in simple, rapid, and sensitive point-of-care (POC) detection.