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.

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.

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 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.

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.

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.

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.

Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA.

Early and accurate diagnosis of viruses is critical for control of the pandemic. CRISPR/Cas-based detection of nucleic acid is an emerging technology for molecular diagnostics, and has been applied for virus detection. Though these methods have excellent sensitivity and specificity, most of them were not able to measure the quantity of virus. We here developed a droplet digital reverse transcription loop-mediated isothermal amplification (RT-LAMP) enhanced Cas12b-based RNA detection platform (RECD), for quantitative detection of viral RNA. CRISPR/Cas12b, which is more thermally stable than other family members in CRISPR systems, is combined with digital RT-LAMP. Due to the innate characteristic of digital format detection and CRISPR/Cas system, droplet digital RECD (ddRECD) assay enables absolute quantification of viral RNA, with single-molecule sensitivity. We expect the ddRECD assay will be a powerful tool for molecular diagnostics.

TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) remains one of the public health problems worldwide. Rapid, sensitive and cost-effective diagnosis of Mycobacterium tuberculosis (M.tb) is critical for TB control. METHODS: We developed a novel M.tb DNA detection platform (nominated as TB-QUICK) which combined loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12b detection. TB-QUICK was performed on pulmonary or plasma samples collected from 138 pulmonary TB (PTB) patients, 21 non-TB patients and 61 close contacts to TB patients. Acid-fast bacillus (AFB) smear, M.tb culture and GeneXpert MTB/RIF (Xpert) assays were routinely conducted in parallel. RESULTS: By targeting M.tb IS6110, TB-QUICK platform could detect as low as 1.3 copy/µL M.tb DNA within 2 h. In pulmonary TB samples, TB-QUICK exhibited improved overall sensitivity of 86.8% over M.tb culture (66.7%) and Xpert (70.4%), with the specificity of 95.2%. More significantly, TB-QUICK exhibited a superior sensitivity in AFB-negative samples (80.5%) compared to Xpert (57.1%) and M.tb culture (46.2%). In the detection of plasma M.tb DNA by TB-QUICK, 41.2% sensitivity for AFB-positive and 31.7% for AFB-negative patients were achieved. CONCLUSION: In conclusion, TB-QUICK exhibits rapidity and sensitivity for M.tb DNA detection with the superiority in smear-negative paucibacillary TB patients. The clinical application of TB-QUICK in TB diagnosis needs to be further validated in larger cohort.

Rapid and Accurate Campylobacter jejuni Detection With CRISPR-Cas12b Based on Newly Identified Campylobacter jejuni-Specific and -Conserved Genomic Signatures.

Campylobacter jejuni is among the most prevalent foodborne zoonotic pathogens leading to diarrheal diseases. In this study, we developed a CRISPR-Cas12b-based system to rapidly and accurately detect C. jejuni contamination. Identification of C. jejuni-specific and -conserved genomic signatures is a fundamental step in development of the detection system. By comparing C. jejuni genome sequences with those of the closely related Campylobacter coli, followed by comprehensive online BLAST searches, a 20-bp C. jejuni-conserved (identical in 1024 out of 1037 analyzed C. jejuni genome sequences) and -specific (no identical sequence detected in non-C. jejuni strains) sequence was identified and the system was then assembled. In further experiments, strong green fluorescence was observed only when C. jejuni DNA was present in the system, highlighting the specificity of this system. The assay, with a sample-to-answer time of ∼40 min, positively detected chicken samples that were contaminated with a dose of approximately 10 CFU C. jejuni per gram of chicken, which was >10 times more sensitive than the traditional Campylobacter isolation method, suggesting that this method shows promise for onsite C. jejuni detection. This study provides an example of bioinformatics-guided CRISPR-Cas12b-based detection system development for rapid and accurate onsite pathogen detection.

The application of a heat-inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants.

The CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low-frequency off-target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium-mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off-target mutation was detected by whole-genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.

A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (Kcat/Km (s-1 M-1) = 8.80 × 1011), which is significantly higher than that of AacCas12b (Kcat/Km (s-1 M-1) = 7.56 × 108) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.