Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

Zhang, Jian; Lv, Hailong; Li, Linxian; Chen, Minjie; Gu, Dayong; Wang, Jin; Xu, Yong

doi:10.3389/fmicb.2021.751408

Cited by 23 publications

(28 citation statements)

References 45 publications

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“…Although CRISPR-based tools take on powerful and robust signal amplification peculiarity, their detection ability is still not enough for directly measure clinical samples without nucleic acid amplification. , To further boost the detection sensitivity of this system, CRISPR technology is usually combined with nucleic acid amplification technology . In particular, when a CRISPR/Cas system was integrated with LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), and other isothermal amplification technologies, nucleic acid detection displayed the features of ease of use, quick turnaround time, and field test capacity. , Some early developed nucleic acid amplification based CRISRP detection methods, such as the SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) technology based on Cas13 nuclease and the DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) and HOLMES (one-Hour Low-cost Multipurpose highly Efficient System) technologies based on Cas12 nuclease, ,− usually contained two separate procedures: nucleic acid amplification and CRISPR end point detection.…”

Section: Introductionmentioning

confidence: 99%

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

et al. 2022

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CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.

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Section: Introductionmentioning

confidence: 99%

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

et al. 2022

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“…Therefore, employing a nucleic acid amplification-free strategy to enhance the detection signal is a promising alternative pathway. 7–9…”

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confidence: 99%

“…Therefore, employing a nucleic acid amplification-free strategy to enhance the detection signal is a promising alternative pathway. [7][8][9] Recently, clustered regularly interspaced short palindromic repeats (CRISPR) as an emerging gene-editing toolbox has received widespread attention. 10,11 Furthermore, diagnostic technologies based on the CRISPR-associated nuclease (Cas) system have also been developed rapidly.…”

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confidence: 99%

Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids

Zhou

Liu

et al. 2022

Chem. Commun.

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“…Платформа характеризуется как точный и простой метод детекции целевых последовательнос тей. При этом преамплификация не требуется [14,51].…”

Section: платформы диагностики на основе систем Crispr/cas V типаunclassified

CRISPR/Cas-based diagnostic platforms

Volkov

Dolgova

Dedkov

2022

Russian Journal of Infection and Immunity

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Over the last few years, CRISPR/Cas systems have been extensively studied and used for a wide range of applied purposes. The variety of their applications is accounted for by the ability of Cas-type nucleases to targetly cleave specified nucleic acid sequences. In this case, the researcher might set the necessary sequence of the guiding elements in the CRISPR/Cas system, played by so-called single guide RNAs allowing it to act on select targets. This potential underlies one of the reasons for exerting interest in CRISPR/Cas systems. One of the first areas for applying these systems was its use for genomic editing. Later, the list of potential opportunities has been expanded: e.g., they can be used in gene therapy and epigenetic research. It is possible to create sgRNA libraries which might be used to create a pool of viral vectors applied for bacterial cell transformation with subsequent cas-protein transduction that cause target gene knockout. This approach allows finding genes responsible for resistance or sensibility to diverse substances. Using such systems in molecular diagnostics of infectious diseases is considered as one of the most promising directions allowing to detect even extremely low concentrations of pathogenic organisms in samples due to their specific nucleotide sequences. Simultaneously, such assays turn out to be accurate, rapid and easy to utilize. In addition, some platforms may work without using expensive equipment, because methods for fast and simple sample preparation have already been developed, whereas modern preamplification approaches allow to avoid applying thermocycling devices. Interestingly, a great amount of diverse types of natural CRISPR/Cas systems have been already discovered. Such abundance promotes development of multiple artificial systems, each of which exerting own unique characteristics. Therefore, a variety of diagnostic platforms with different properties are created on their basis that allows researchers and physicians to choose an optimal approach for performing specific tasks. For this reason, insights into structure and operation of CRISPR/Cas systems are necessary for selecting a suitable platform. The current classification of systems is based on such principles serving as the basis, in turn, for convenient evaluation of the very variety of molecular diagnostics platforms and presentation of the typical technical characteristics and nuances for each method. Thus, this review, which is mainly devoted to the platforms for molecular diagnostics of infectious diseases, also touches upon the issues of functioning, devices, and classification of CRISPR/Cas systems.

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Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

Cited by 23 publications

References 45 publications

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids

CRISPR/Cas-based diagnostic platforms

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