A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

Nguyễn, Long Thanh; Macaluso, Nicolas C.; Pizzano, Brianna L.M.; Cash, Melanie N.; Spacek, Jan; Karasek, Jan; Miller, Megan R.; Lednicky, John A.; Dinglasan, Rhoel R.; Salemi, Marco; Jain, Piyush

doi:10.1016/j.ebiom.2022.103926

Cited by 60 publications

(76 citation statements)

References 28 publications

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“…Upon amplification, correct base-pairing between the guide RNA and the amplified target serves as an additional checkpoint to provide accurate signal readouts. The one-pot reaction further eliminates carryover contamination that is frequently observed with two-pot RT-LAMP-coupled platforms 10 . Most RT-LAMP reactions work optimally between 60°C – 65°C 7, 11 ; however, only a handful of Cas enzymes are functional at this temperature range 6, 34 .…”

Section: Resultsmentioning

confidence: 99%

“…If paired with a more rapid extraction method, the overall assay duration can be reduced. Additionally, one-pot reagents can potentially be lyophilized as we discussed in our previous study, thus allowing for administration in low-resource areas 10 . We believe that the SPLENDID system has wide applications for the detection of emerging infectious diseases and can be quickly deployed to regions of interest at a low cost.…”

Section: Discussionmentioning

confidence: 99%

“…SYP-B805 (BrCas12b) and exhibited high enzymatic activity up to 63.4 o C within a single-pot CRISPR-diagnostic assay for the detection of nucleic acids. Our one-pot assay successfully distinguished multiple SARS-CoV-2 Variants of Concerns (VOCs) with high sensitivity and specificity within 20 minutes 10 . However, while BrCas12b allowed for detection at a higher temperature than AapCas12b, the range of detection is still narrow.…”

Section: Introductionmentioning

confidence: 99%

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Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

Nguyễn

Rananaware

Yang

et al. 2022

Preprint

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics have elevated nucleic acid detection in terms of sensitivity, specificity, and rapidity in recent years. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify workflow and reduce carryover contamination. Here, we report an engineered Cas12b system from Brevibacillus (eBrCas12b) with improved thermostability that falls within the optimal range (60-65°C) of the Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP). Using de novo structural analyses via DeepDDG and HotSpot Wizard based on Alpha Fold and SWISS-MODEL predicted structures, mutations were introduced into the REC and RuvC domains of wild-type BrCas12b to tighten the hydrophobic cores of the protein, thereby enhancing its stability at high temperatures. We expressed, purified, and systematically characterized 49 BrCas12b variants with the emphasis on functionality and thermostability. The assay utilizing eBrCas12b, which we coined 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-4°C and 7°C higher than wild-type BrCas12b and AapCas12b, respectively. We further validated SPLENDID clinically in 40 Hepatitis C (HCV) positive and 40 negative serum samples. A specificity of 97.5%, an accuracy of 90.0%, and a sensitivity of 82.5% were achieved. Results can be obtained via one-pot testing in as little as 20 minutes. With the extraction process, the entire assay can be performed in under an hour. Therefore, we believe that SPLENDID has the potential to become a widely universal platform for the detection of infectious diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

Nguyễn

Rananaware

Yang

et al. 2022

Preprint

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“…CRISPR has already been used to detect malaria strain variants in mixed infections, an important challenge for malaria control and elimination efforts and a modality that could be extended to many areas of epidemiology ( Kwiatkowski, 2015 ⁠. One-pot systems that are suitable for field deployment due to their minimal technological and logistical prerequisites, high diagnostic reliability, and rapid turnaround time have been developed for SARS-CoV-2 and show additional promise for the expansion of these tools in malaria-endemic countries ( Nguyen et al., 2021 ). The development of isothermal reactions performed with lyophilized reagents and with straightforward visualization at the point-of-care are particularly revolutionary as a means to quickly, cheaply, and accurately diagnose malaria infections in low-resource and/or remote locations ( Cunningham et al., 2021 ).…”

Section: Overviewmentioning

confidence: 99%

Potential Opportunities and Challenges of Deploying Next Generation Sequencing and CRISPR-Cas Systems to Support Diagnostics and Surveillance Towards Malaria Control and Elimination in Africa

Lyimo

Popkin-Hall

Giesbrecht

et al. 2022

Front. Cell. Infect. Microbiol.

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Recent developments in molecular biology and genomics have revolutionized biology and medicine mainly in the developed world. The application of next generation sequencing (NGS) and CRISPR-Cas tools is now poised to support endemic countries in the detection, monitoring and control of endemic diseases and future epidemics, as well as with emerging and re-emerging pathogens. Most low and middle income countries (LMICs) with the highest burden of infectious diseases still largely lack the capacity to generate and perform bioinformatic analysis of genomic data. These countries have also not deployed tools based on CRISPR-Cas technologies. For LMICs including Tanzania, it is critical to focus not only on the process of generation and analysis of data generated using such tools, but also on the utilization of the findings for policy and decision making. Here we discuss the promise and challenges of NGS and CRISPR-Cas in the context of malaria as Africa moves towards malaria elimination. These innovative tools are urgently needed to strengthen the current diagnostic and surveillance systems. We discuss ongoing efforts to deploy these tools for malaria detection and molecular surveillance highlighting potential opportunities presented by these innovative technologies as well as challenges in adopting them. Their deployment will also offer an opportunity to broadly build in-country capacity in pathogen genomics and bioinformatics, and to effectively engage with multiple stakeholders as well as policy makers, overcoming current workforce and infrastructure challenges. Overall, these ongoing initiatives will build the malaria molecular surveillance capacity of African researchers and their institutions, and allow them to generate genomics data and perform bioinformatics analysis in-country in order to provide critical information that will be used for real-time policy and decision-making to support malaria elimination on the continent.

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“…First, we conducted a search in PubMed with "qPCR diagnosis COVID19" as keywords and compared the output with the number of publications on RT-LAMP and RT-LAMP-CRISPR strategies for COVID-19 diagnosis [15]. Finally, we collected data on the different sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) from 10 papers related to RT-LAMP and 10 papers on the RT-LAMP-CRISPR-Cas COVID19 diagnostic technique [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. We used the results to calculate the parameters needed for the comparison.…”

Section: Study Of the State Of The Artmentioning

confidence: 99%

RT-LAMP-CRISPR-Cas13a technology as a promising diagnostic tool for the SARS-CoV-2 virus

Ortiz-Cartagena

Fernández‐García

Blasco

et al. 2022

Preprint

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At the end of 2019, the new coronavirus, SARS-CoV-2, began a pandemic that persists to date and which has caused more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development in diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays a collateral activity against single stranded RNA molecules. With the aim of improve the sensitivity of the diagnosis, this technology is usually combined with isothermal pre-amplification reactions (SHERLOCK, DETECTR). Basing on this, we have developed an RT-LAMP-CRISPR-Cas13a for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction kit that exhibited 100 % specificity and 83 % sensitivity, as well as a positive predictive value of 100 % and a negative predictive value of 100%, 81%, 79.1% and 66.7 % in <20 Ct, 20-30 Ct, >30 Ct and total Ct values, respectively.

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A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

Cited by 60 publications

References 28 publications

Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

Engineering Highly Thermostable Cas12b via De Novo Structural Analyses for One-Pot Detection of Nucleic Acids

Potential Opportunities and Challenges of Deploying Next Generation Sequencing and CRISPR-Cas Systems to Support Diagnostics and Surveillance Towards Malaria Control and Elimination in Africa

RT-LAMP-CRISPR-Cas13a technology as a promising diagnostic tool for the SARS-CoV-2 virus

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