The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

CRISPR-Mediated Viral Gene Knockout to Investigate Viral Evasion of Antiviral Innate Immunity.

The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.

CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

One-Pot Assay for Rapid Detection of Stenotrophomonas maltophilia by RPA-CRISPR/Cas12a.

Stenotrophomonas maltophilia (S. maltophilia, SMA) is a common opportunistic pathogen that poses a serious threat to the food industry and human health. Traditional detection methods for SMA are time-consuming, have low detection rates, require complex and expensive equipment and professional technical personnel for operation, and are unsuitable for on-site detection. Therefore, establishing an efficient on-site detection method has great significance in formulating appropriate treatment strategies and ensuring food safety. In the present study, a rapid one-pot detection method was established for SMA using a combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a, referred to as ORCas12a-SMA (one-pot RPA-CRISPR/Cas12a platform). In the ORCas12a-SMA detection method, all components were added into a single tube simultaneously to achieve one-pot detection and address the problems of nucleic acid cross-contamination and reduced sensitivity caused by frequent cap opening during stepwise detection. The ORCas12a-SMA method could detect at least 3 × 10° copies·µL-1 of SMA genomic DNA within 30 min at 37 °C. Additionally, this method exhibited sensitivity compared to the typical two-step RPA-CRISPR/Cas12a method. Overall, the ORCas12a-SMA detection offered the advantages of rapidity, simplicity, high sensitivity and specificity, and decreased need for complex large-scale instrumentation. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in SMA detection and is highly suitable for point-of-care testing. It helps reduce losses in the food industry and provides assistance in formulating timely and appropriate antimicrobial treatment plans.

An ultra-sensitive suboptimal protospacer adjacent motif enhanced rolling circle amplification assay based on CRISPR/Cas12a for detection of miR-183.

Introduction: MicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable upregulation in individuals with the condition. However, the development of highly sensitive miRNAs assays for the rapid diagnosis of DR in clinical settings remains a challenging task. Methods: In this study, we introduce an enhanced CRISPR/Cas12a assay, leveraging suboptimal PAM (sPAM)-mediated Cas12a trans-cleavage in conjunction with rolling circle amplification (RCA). sPAM was found to perform better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations and was used instead of canonical PAM (cPAM) to mediate the detection. The parameters of reactions have also been optimized. Results and discussion: In comparison with cPAM, sPAM has higher sensitivity in the detection of ssDNA at concentrations lower than 10 pM by Cas12a. By replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity for miR-183 detection is achieved, with a detection limit of 0.40 aM. within 25 min and a linear range spanning from 1 aM. to 1 pM. Our assay also exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples is also validated. This study introduces a groundbreaking assay with excellent performance through a simple modification, which not only addresses existing diagnostic challenges, but also opens exciting new avenues for clinical diagnosis in the realm of DR.

CRISPR/Cas9 in the treatment of sickle cell disease (SCD) and its comparison with traditional treatment approaches: a review.

Sickle cell disease (SCD) is a common hereditary blood disorder that profoundly impacts individuals' health, causing chronic pain, anemia, organ damage, increased susceptibility to infections, and social and psychological effects. Over the years, advances in treatment have improved the long-term outcomes of SCD patients. However, problems such as limited access to hematopoietic stem cell transplantation (HSCT) and potential complications associated with the available therapies underscore the importance of continued research and development. The recent FDA approval of Casgevy (Exagamglogene autotemcel), a genetic therapy based on CRISPR/Cas9 technology, demonstrates a comprehensive effort to address the complexity of SCD using new technologies. This review explores the potential of CRISPR/Cas9 for treating SCD and evaluates its efficacy, safety, and long-term outcomes compared to traditional treatment approaches. Long-term research is needed to comprehensively assess the safety, effectiveness, and inclusion of CRISPR/Cas9, ensuring its overall efficacy.

A rapid visualization method for detecting rotavirus A by combining nuclear acid sequence-based amplification with the CRISPR-Cas12a assay.

Introduction. Rotavirus A is the most common pathogen causing diarrhoea in children less than 5 years, leading to severe complications such as dehydration, electrolyte imbalances, acidosis, myocarditis, convulsions, pneumonia, and other life-threatening conditions.Gap statement. There is an urgent need for a rapid and efficient nucleic acid detection strategy to enable early diagnosis and treatment, preventing rotavirus transmission and associated complications.Aim. This article aimed to develop a nuclear acid sequence-based amplification (NASBA)-Cas12a system for detecting rotavirus A using fluorescence intensity or lateral flow strips.Methodology. The NASBA technology was combined with the clustered regularly interspaced short palindromic repeats-Cas12a system to establish a NASBA-Cas12a system for detecting rotavirus A.Results. The NASBA-Cas12a system could detect rotavirus A at 37 ℃ within 70 min and had no cross-reactivity with other viruses, achieving a limit of detection of 1.2 copies µl-1. This system demonstrated a sensitivity of 100%, specificity of 90%, positive predictive value of 97.22% and negative predictive value of 100%. The kappa value was 0.933, indicating that the NASBA-Cas12a system was highly consistent with reverse transcription-PCR.Conclusion. The NASBA-Cas12a system exhibited high sensitivity and specificity for detecting rotavirus A, showing great potential for clinical application.

LAMP-CRISPR/Cas12a-based impedimetric biosensor powered by Fe3O4@Au-(S-polyA-S)-Au for detection of SARS-CoV-2.

A low-cost, lab-made polytetrafluoroethylene micro-cell, equipped with three electrodes, wasd eveloped for the impedimetric detection of SARS-CoV-2. The gold working electrode was modified with a double-ended thiolated poly-adenine probe, which was conjugated with magnetic Fe3O4@Au nanoparticles (Fe3O4@Au-(S-polyA-S)-Au). After the loop-mediated isothermal amplification (LAMP) of viral RNA, the single-guide RNA (sgRNA), specifically bound to the SARS-CoV-2 target sequence, activates Cas12a. Cas12a then cleaved the immobilized probe. As a result, the magnetic Fe3O4@Au nanoparticles were released and adsorbed onto the gold electrode surface, using an external magnet. This process increased the physical surface area of the gold electrode, facilitating redox ion ([FeIII/II(CN)6]3-/4-) electron transfer. The decrease in the charge transfer resistance was utilized for SARS-CoV-2 detection. Our LAMP-CRISPR/Cas12a-based impedimetric biosensor, powered by Fe3O4@Au-(S-polyA-S)-Au, demonstrated impressive capabilities, including a remarkable detection limit of 0.8 aM (0.48 copies/µL) and a linear range of 0.01 to 36.06 fM.

GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida.

Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse Pseudomonas putida that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.

N318L Blocks the Interaction of Fluralaner but Not Broflanilide or Fipronil with the Insect GABA Receptor In Vivo.

Fluralaner is a novel insecticide targeting the ionotropic GABA receptor (GABAR) subunit, RDL. A recent study revealed that N316L, a substitution of asparagine (N) with leucine (L), in the second transmembrane (M2)-spanning region reduced the antagonist action of fluralaner on the housefly Musca domestica RDL (MdRDL) in vitro. To verify the impact of N316L in vivo, the corresponding mutation (N318L) in the fruitfly Drosophila melanogaster RDL (DmRDL) was constructed using CRISPR/Cas9 genome editing. The homozygous DmRDLN318L mutant showed a 9.87-fold resistance to fluralaner compared with w1118 while still being highly sensitive to broflanilide and fipronil, which is consistent with those findings observed in the electrophysiology assays of the homomeric DmRDLWT or DmRDLN318L channel. Moreover, DmRDLN318L led to malformed ovaries, stunted eggs, and sterility in homozygous females. These results highlighted N318 as a molecular site for fluralaner in vivo and in vitro and might elucidate the resistance mechanisms of insects against fluralaner.

Raman-enhanced sensor based on CRISPR-SERS technology for the rapid and hypersensitive detection of Mycobacterium tuberculosis.

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/µL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.

Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.

BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.

[Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster].

An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.

Cotton Bollworm (H. armigera) Effector PPI5 Targets FKBP17-2 to Inhibit ER Immunity and JA/SA Responses, Enhancing Insect Feeding.

The cotton bollworm causes severe mechanical damage to plants during feeding and leaves oral secretions (OSs) at the mechanical wounds. The role these OSs play in the invasion of plants is still largely unknown. Here, a novel H. armigera effector peptidyl prolyl trans-isomerase 5 (PPI5) was isolated and characterized. PPI5 induces the programmed cell death (PCD) due to the unfolded protein response (UPR) in tobacco leaf. We reveal that PPI5 is important for the growth and development of cotton bollworm on plants, as it renders plants more susceptible to feeding. The GhFKBP17-2, was identified as a host target for PPI5 with peptidyl-prolyl isomerase (PPIase) activity. CRISPR/Cas9 knock-out cotton mutant (CR-GhFKBP17-1/3), VIGS (TRV: GhFKBP17-2) and overexpression lines (OE-GhFKBP17-1/3) were created and the data indicate that GhFKBP17-2 positively regulates endoplasmic reticulum (ER) stress-mediated plant immunity in response to cotton bollworm infestation. We further confirm that PPI5 represses JA and SA levels by downregulating the expression of JA- and SA-associated genes, including JAZ3/9, MYC2/3, JAR4, PR4, LSD1, PAD4, ICS1 and PR1/5. Taken together, our results reveal that PPI5 reduces plant defense responses and makes plants more susceptible to cotton bollworm infection by targeting and suppressing GhFKBP17-2 -mediated plant immunity.

A genome-wide CRISPR/Cas9 knockout screen identifies SEMA3F gene for resistance to cyclin-dependent kinase 4 and 6 inhibitors in breast cancer.

BACKGROUND: Palbociclib is a cell-cycle targeted small molecule agent used as one of the standards of care in combination with endocrine therapy for patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. Although several gene alterations such as loss of Rb gene and amplification of p16 gene are known to be conventional resistance mechanisms to cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, the comprehensive landscape of resistance is not yet fully elucidated. The purpose of this study is to identify the novel resistant genes to the CDK4/6 inhibitors in HR-positive HER2-negative breast cancer. METHODS: The whole genome knockout screen using CRISPR/Cas9 genome editing was conducted in MCF7 to identify resistant genes to palbociclib. The candidate genes for resistance were selected by NGS analysis and GSEA analysis and validated by cell viability assay and mouse xenograft models. RESULTS: We identified eight genes including RET, TIRAP, GNRH1, SEMA3F, SEMA5A, GATA4, NOD1, SSTR1 as candidate genes from the whole genome knockout screen. Among those, knockdown of SEMA3F by siRNA significantly and consistently increased the cell viability in the presence of CDK4/6 inhibitors in vitro and in vivo. Furthermore, the level of p-Rb was maintained in the palbociclib treated SEMA3F-downregulated cells, indicating that the resistance is driven by increased activity of cyclin kinases. CONCLUSION: Our observation provided the first evidence of SEMA3F as a regulator of sensitivity to CDK4/6 inhibitors in breast cancer. The detailed mechanisms of resistance deserve further functional studies to develop the better strategy to overcome resistance in CDK4/6 inhibitors.

CRISPR/Cas-Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen-Resistant Pulse Crops.

Pulses provide myriad health benefits and are advantageous in an environmental context as a result of their leguminous nature. However, phytopathogenic fungi, oomycetes and bacteria pose a substantial threat to pulse production, at times leading to crop failure. Unfortunately, existing disease management strategies often provide insufficient control, and there is a clear need for the development of new pulse cultivars with durable and broad-spectrum disease resistance. CRISPR/Cas-mediated gene editing has proven its potential for rapidly enhancing disease resistance in many plant species. However, this tool has only very recently been applied in pulse species, and never in the context of plant immunity. In this review, we examine the recent successful utilization of this technology in pulse species for proof-of-concept or the improvement of other traits. In addition, we consider various genes that have been edited in other plant species to reduce susceptibility to pathogens, and discuss current knowledge regarding their roles in pulses. Given the functional conservation of the selected genes across diverse plant species, there is a high likelihood that their editing would elicit similar effects in non-oilseed grain legumes, thus providing a suite of potential targets for CRISPR/Cas-mediated gene editing to promote pulse crop productivity in coming years.

Advanced Electrochemical Biosensing toward Staphylococcus aureus Based on the RPA-CRISPR/Cas12a System and Conductive Nanocomposite.

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating S. aureus. In the presence of S. aureus, the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting S. aureus. The linear range for quantifying S. aureus in pure culture is 1.04 × 101-1.04 × 108 CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of S. aureus in milk within a linear range of 1.07 × 101-1.07 × 107 CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.