Discovery and engineering of ChCas12b for precise genome editing.

Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.

CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic HBV DNA in one-pot.

BACKGROUND: Currently, millions of people suffer from undiagnosed chronic hepatitis B (CHB) infection each year, which leads to high mortality rates attributed to cirrhosis and hepatocellular carcinoma. Previously reported assays, such as PCR-based assays, have limitations in terms of convenient for CHB screening in high-burden regions and resource-limited settings. Recently, diagnosis based on CRISPR/Cas, which has been considered as a potential method of point-of-care test (POCT) in resource-limited settings, offers a significant advantage in terms of high sensitivity and specificity. Therefore, there is an urgent need for the hepatitis B virus (HBV) detection utilizing CRISPR/Cas system. RESULTS: We have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR. SIGNIFICANCE: The utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.

Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4).

The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40 min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering.

Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5-6 bases, corresponding to only 2-3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b-based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new-generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin-resistant E. coli strain (BL21) and a 6.49-fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection.

Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5'-/6-FAM/CCCCCCCC/BHQ1/-3') presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod).

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/µL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction.

IMPORTANCE: In this study, we successfully established a new One-Pot method, named TB One-Pot, for detecting Mtb in sputum by combining CRISPR-cas12b-mediated trans-cleavage with cross-priming amplification (CPA). Our study evaluated the diagnostic performance of TB One-Pot in clinical sputum samples for tuberculosis. The findings provide evidence for the potential of TB One-Pot as a diagnostic tool for tuberculosis.

Engineering of the LAMP-CRISPR/Cas12b platform for Chlamydia psittaci detection.

Introduction. Chlamydia psittaci (C. psittaci) is a zoonotic infection, that causes psittacosis (parrot fever) in humans, leading to severe clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, in rare cases, death.Gap Statement. Rapid, sensitive and specific detection of C. psittaci facilitates timely diagnosis and treatment of patients.Aim. This study aimed to engineer the LAMP-CRISPR/Cas12b platform for C. psittaci detection.Methodology. The loop-mediated isothermal amplification (LAMP) technique and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 12b (CRISPR-Cas12b) assay were combined to establish two-step and one-tube LAMP-CRISPR/Cas12b reaction systems, respectively, for rapidly detecting C. psittaci.Results. The two-step and one-tube LAMP-CRISPR/Cas12b assay could complete detection within 1 h. No cross-reactivity was observed from non-C. psittaci templates with specific LAMP amplification primers and single-guide RNA (sgRNA) targeting the highly conserved short fragment CPSIT_0429 gene of C. psittaci. The detection limits of the two-step and one-tube LAMP-CRISPR/Cas12b reaction were 102 aM and 103 aM, respectively. The results were consistent with qPCR for nucleic acid detection in 160 clinical samples, including 80 suspected C. psittaci samples, kept in the laboratory.Conclusions. The LAMP-CRISPR/Cas12b assay developed in this study provides a sensitive and specific method for rapidly detecting C. psittaci and offers technical support for its rapid diagnosis.

Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform.

Mycoplasma pneumoniae (MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination.

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures.

BACKGROUND: The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a. METHODS: We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells. FINDINGS: We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such "hypermutated" HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation. INTERPRETATION: These results provide in vitro "proof of concept' for CRISPR-Cas12b mediated HIV-1 inactivation.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

CDetection.v2: One-pot assay for the detection of SARS-CoV-2.

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

CRISPR-Cas12-Based Diagnostic Applications in Infectious and Zoonotic Diseases.

Rapid detection of infectious and zoonotic diseases is very important for pathogen identification and infection control. Molecular diagnostic assays are well-known for high accuracy and sensitivity; however, conventional methods such as real-time PCR may require professional instruments and operations, preventing their wide applications in scenarios including animal quarantine. The recently developed CRISPR diagnostic (CRISPR-Dx) methods, employing the trans-cleavage activities of either Cas12 (e.g., HOLMES) or Cas13 (e.g., SHERLOCK), have shown great potential in rapid and convenient nucleic acid detection. Guided by specially designed CRISPR RNA (crRNA), Cas12 binds target DNA sequences and trans-cleaves ssDNA reporters, generating detectable signals, while Cas13 recognizes target ssRNA and trans-cleaves ssRNA reporters. To achieve high detection sensitivity, both HOLMES and SHERLOCK systems can be combined with pre-amplification procedures including both PCR and isothermal amplifications. Here, we present the employment of the HOLMESv2 method for convenient detection of the infectious and zoonotic diseases. Specifically, target nucleic acid is first amplified by LAMP or RT-LAMP, and the products are then detected by the thermophilic Cas12b. In addition, Cas12b reaction can be combined with LAMP amplification to achieve one-pot reaction systems. In this chapter, we provide a step-by-step description of the HOLMESv2-mediated rapid and sensitive detection of Japanese encephalitis virus (JEV), an RNA pathogen as an example.

Development of CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings.

Tuberculosis (TB) is a chronic infectious disease with high mortality caused by the Mycobacterium tuberculosis complex (MTC). Its clinical symptoms include a prolonged cough with mucus, pleuritic chest pain, hemoptysis, etc., and predominant complications such as tuberculous meningitis and pleural effusion. Thus, developing rapid, ultrasensitive, and highly specific detection techniques plays an important role in controlling TB. Here, we devised CRISPR/CRISPR-associated 12b nuclease (CRISPR/Cas12b)-based multiple cross displacement amplification technique (CRISPR-MCDA) targeting the IS6110 sequence and used it to detect MTC pathogens. A newly engineered protospacer adjacent motif (PAM) site (TTTC) was modified in the linker region of the CP1 primer. In the CRISPR-MCDA system, the exponentially amplified MCDA amplicons with the PAM sites can guide the Cas12b/gRNA complex to quickly and accurately recognize its target regions, which successfully activates the CRISPR/Cas12b effector and enables ultrafast trans-cleavage of single-stranded DNA reporter molecules. The limit of detection of the CRISPR-MCDA assay was 5 fg/µL of genomic DNA extracted from the MTB reference strain H37Rv. The CRISPR-MCDA assay successfully detected all examined MTC strains and there was no cross-reaction with non-MTC pathogens, confirming that its specificity is 100%. The entire detection process can be completed within 70 min using real-time fluorescence analysis. Moreover, visualization detection (under UV light) was also designed to verify the results, eliminating the use of specialized instruments. In conclusion, the CRISPR-MCDA assay established in this report can be used as a valuable detection technique for MTC infection. IMPORTANCE The Mycobacterium tuberculosis complex pathogen is a crucial infectious agent of tuberculosis. Hence, improving the capability of MTC detection is one of the most urgently required strategies for preventing and controlling TB. In this report, we successfully developed and implemented CRISPR/Cas12b-based multiple cross displacement amplification targeting the IS6110 sequence to detect MTC pathogens. These results demonstrated that the CRISPR-MCDA assay developed in this study was a rapid, ultrasensitive, highly specific, and readily available method which can be used as a valuable diagnostic tool for MTC infection in clinical settings.

Ultrasensitive and Specific Identification of Monkeypox Virus Congo Basin and West African Strains Using a CRISPR/Cas12b-Based Platform.

Human monkeypox (MPX) is a severe and reemerging infectious disease caused by monkeypox virus (MPXV) and forms two distinct lineages, including Congo Basin and West African clades. Due to the absence of specific vaccines and antiviral drugs, developing a point-of-care (POC) testing system to identify MPXV is critical for preventing and controlling MPX transmission. Here, a CRISPR/Cas12b diagnostic platform was integrated with loop-mediated isothermal amplification (LAMP) to devise a novel CRISPR-MPXV approach for ultrasensitive, highly specific, rapid, and simple detection of MPXV Congo Basin and West African strains, and the detection results were interpreted with real-time fluorescence and a gold nanoparticle-based lateral flow biosensor (AuNP-LFB). The optimal detection process, including genomic DNA extraction (15 min), LAMP preamplification (35 min at 66°C), CRISPR/Cas12b-based detection (5 min at 45°C), and AuNP-LFB readout (~2 min), can be completed within 60 min without expensive instruments. Our assay has a limit of detection of 10 copies per test and produces no cross-reaction with any other types of pathogens. Hence, our CRISPR-MPXV assay exhibited considerable potential for POC testing for identifying and distinguishing MPXV Congo Basin and West African strains, especially in regions with resource shortages. IMPORTANCE Monkeypox (MPX), a reemerging zoonotic disease caused by monkeypox virus (MPXV), causes a smallpox-like disease in humans. Early diagnosis is critical to prevent MPX epidemics. Here, CRISPR/Cas12b was integrated with LAMP amplification to devise a novel CRISPR-MPXV approach to achieve highly specific, ultrasensitive, rapid, and visual identification of MPXV Congo Basin and West African strains.

An extraction-free one-step CRISPR-assisted detection platform and a potential Streptococcus pneumoniae at-home self-testing kit.

A rapid, accurate, easy-to-use nucleic acid detection technology is essential for disease diagnosis and control. Herein, we improved CRISPR-top (cluster regularly interspaced short palindromic repeats-mediated testing in one-pot) to develop Extraction-free one-step CRISPR-assistant detection (ExCad), a simple, rapid, accurate gene detection tool for unextracted colonies and samples. We established a pretreatment protocol to rapidly liquify sputum samples and release nucleic acids within 10 min. The ExCad results can be visualised by a real-time fluorescence reader or the naked eye under blue light. We developed an ExCad-Sp assay to detect Streptococcus pneumoniae from unextracted strains and specimens, and optimised the assay conditions. Assay feasibility was evaluated using sputum samples from 32 patients, and it achieved 92.9 % (13/14) sensitivity, 100 % (18/18) specificity, 100 % (13/13) positive predictive value, and 94.7 % (18/19) negative predictive value compared with bacteria culture. The ExCad-Sp assay has potential for developing an at-home self-testing kit for S. pneumoniae.

Accurate, Sensitive, and Rapid Detection of Pseudomonas aeruginosa Based on CRISPR/Cas12b with One Fluid-Handling Step.

Pseudomonas aeruginosa is a major bacterial pathogen causing nosocomial infections and accounts for morbidity and mortality among patients with cystic fibrosis. An accurate, sensitive, and rapid method to detect P. aeruginosa is critical for the early control of infection and patient management. In this study, we established a P. aeruginosa clustered regularly interspaced short palindromic repeats testing in one pot (CRISPR-top) assay which detected P. aeruginosa with one fluid-handling step in one tube. The reaction was performed isothermally within 1 h; thus, specific instruments were not required. The optimal reaction conditions of this assay were determined to be a temperature of 55°C; working concentrations of 1 µM for the forward inner primer and backward inner primer, 0.5 µM for the loop forward primer and loop backward primer, and 0.25 µM for the forward outer primer and backward outer primer; as well as a 2 µM concentration single-stranded DNA reporter molecules. In terms of specificity, our assay showed 100% inclusivity and exclusivity among 48 strains, including 15 P. aeruginosa clinical isolates and 33 non-P. aeruginosa strains. The limit of detection of our method was 10 copies per reaction mixture. Forty-six human sputum specimens from patients with respiratory symptoms were tested. Using the results of quantitative real-time PCR as the gold standard, our method showed 85.3% (29/34) sensitivity, 100% (12/12) specificity, a positive predictive value of 100% (29/29), and a negative predictive value of 70.6% (12/17). In summary, the P. aeruginosa CRISPR-top assay developed in the present study is a high-efficiency alternative tool for the accurate and rapid detection of P. aeruginosa, especially in resource-limited settings. IMPORTANCE This study reports a P. aeruginosa CRISPR-top assay which can precisely identify P. aeruginosa using nucleic acids from pure cultures or clinical samples in one pot with one fluid-handling step. The P. aeruginosa CRISPR-top reaction is suitable for on-site testing, and its diagnostic performance can be compared with that of qPCR.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

One-Pot Isothermal LAMP-CRISPR-Based Assay for Klebsiella pneumoniae Detection.

Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens causing nosocomial infection. A rapid, accurate, and convenient detection method is required for early diagnosis and directed therapy of K. pneumoniae infection. CRISPR-top (CRISPR-mediated testing in one pot) is a LAMP-CRISPR-based nucleic acid detection platform, which integrates target preamplification with CRISPR/Cas12b-based detection into a one-pot reaction mixture, performed at a constant temperature. In this study, we established the K. pneumoniae CRISPR-top assay to precisely identify K. pneumoniae at 56°C within 60 min. The reaction mixture with 0.53 µM (each) FIP and BIP, 0.27 µM LF, 0.13 µM (each) F3 and B3, and 2 µM ssDNA fluorescence probe was determined as the optimal reaction system of our assay. The limit of detection of this assay is 1 pg genomic DNA (equivalent to 160 K. pneumoniae cells and 1.6 × 105 CFU/mL for samples) per reaction, which is 10-fold more sensitive than LAMP. Up to 105 strains composed of K. pneumoniae clinical isolates and non-K. pneumoniae strains were correctly identified by our assay. A total of 58 sputum samples collected from patients with respiratory symptoms were used to evaluate the diagnostic performance of the K. pneumoniae CRISPR-top assay. As a result, the K. pneumoniae CRISPR-top assay yielded 100% (33/33) specificity and 96% (24/25) sensitivity, as well as a positive predictive value of 100% (24/24) and a negative predictive value of 97.1% (33/34), which were all higher than LAMP detection. In conclusion, the K. pneumoniae CRISPR-top assay developed in this study is a simple, rapid and ultra-specific method to detect K. pneumoniae. IMPORTANCE Klebsiella pneumoniae is a significant threat to global health. At present, the methods of K. pneumoniae detection are culture-based and instrument-dependent and are not suitable for rapid diagnostic. This study reports K. pneumoniae CRISPR-top assay, which can precisely identify K. pneumoniae using nucleic acids of pure cultures or clinical samples in one pot with one fluid-handling step. The K. pneumoniae CRISPR-top reaction can be completed within 60 min at a constant temperature, thus specific instruments are not required. Our results show that CRISPR-top assay yields enormous advantages compared with LAMP detection. The K. pneumoniae CRISPR-top assay can be a high-efficiency alternative tool for rapid and accurate diagnosis of K. pneumoniae infection, especially in resource-limited settings.