Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains

Zhang, Yihao; Qian, Long; Wei, Weijia; Wang, Yu; Wang, Beining; Lin, Pingping; Li, Wenchao; Xu, Luze; Li, Xiang; Liu, Dongming; Cheng, Sida; Li, Jiaofeng; Ye, Yixuan; Li, Hang; Zhang, Xiaohan; Dong, Yun; Zhao, Xuejin; Liu, Cuihua; Zhang, Haoqian; Ouyang, Qi; Lou, Chunbo

doi:10.1021/acssynbio.6b00215

Cited by 144 publications

(122 citation statements)

References 23 publications

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“…Over the last decade, considerable efforts have been devoted to engineering Cas nucleases to construct novel nucleic acid detection platforms, such as the dCas9‐split horseradish peroxidase (HRP) fusion protein (Qiu et al, 2018) and the dCas9‐split‐luciferase fusion protein (Y. Zhang et al, 2017). Recently, the Li group constructed an mRNA‐sensing platform by engineering a conditional sgRNA, rather than a Cas9 protein (Figure 4a; Y. Li, Teng, Zhang, Deng, & Li, 2019).…”

Section: Detection Of Rnamentioning

confidence: 99%

“…Inspiringly, researchers found that accurate detection of live Mtb, the causative agent of TB, could be an alternative approach for TB diagnosis (Y. Chen et al, 2018). Following this finding, a powerful in vitro Mtb detection platform was built based on dCas9 and split‐luciferase technology, termed the Paired dCas9 (PC) reporter system (Figure 7b; Y. Zhang et al, 2017). Specifically, a pair of split‐luciferase reporter‐linked dCas9 proteins was programmed to target the adjacent sequences of the Mtb genome with the help of specific sgRNAs.…”

Section: Detection Of Pathogenic Bacteriamentioning

confidence: 99%

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Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

Zhu

Liu

Xie

et al. 2020

Biotech & Bioengineering

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Beyond their widespread application as genome‐editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR‐Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR‐Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR‐based detection methods will be discussed according to the most recent developments.

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Section: Detection Of Rnamentioning

confidence: 99%

Section: Detection Of Pathogenic Bacteriamentioning

confidence: 99%

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

Zhu

Liu

Xie

et al. 2020

Biotech & Bioengineering

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“…(2017) created a diagnostic test based on two dCas9 proteins fused with split domains of luciferase enzyme 51 . Binding of two dCas9 proteins to the adjacent target DNA results in re-constitution of luciferase and emission of luminescent signal that can be readily detected by a luminometer.…”

Section: Detection Of Nucleic Acids By Crispr-cas Type II Systemsmentioning

confidence: 99%

“…Binding of two dCas9 proteins to the adjacent target DNA results in re-constitution of luciferase and emission of luminescent signal that can be readily detected by a luminometer. As a proof of concept, this technology was shown to detect Mycobacterium tuberculosis with high specificity and sensitivity 51 .…”

Section: Detection Of Nucleic Acids By Crispr-cas Type II Systemsmentioning

confidence: 99%

CRISPR-Cas Systems for Diagnosing Infectious Diseases

Kostyusheva¹,

Brezgin²,

Babin³

et al. 2020

Preprint

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Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas9, Cas12, Cas13, Cas14) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, or lateral flow assay detection. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing coronoviral nCov-2019 infection) urgently need the developing of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

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“…Several DNA detection methods were developed by using the Cas9 nickase activity, such as Cas9nAR (Cas9 nickase-based amplification reaction) (19), and CRISDA (CRISPR-Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification) (20). In addition, several DNA detection methods were developed by using the catalytically deactivated Cas9 (dCas9), such as CRISPR-Chip (21) and paired dCas9 proteins linked to split halves of luciferase (22). However, the potential of Cas9/sgRNA system in nucleic acid detection and typing has not yet been fully explored by the current studies.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique

Luo

Gao

et al. 2020

Preprint

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Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present COVID-19 pandemic. The simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, the current nucleic acid detection techniques are still limited the traditional amplification and hybridization. To overcome the limitation, we here develop a CRISPR/Cas9-assisted DNA detection (CADD). In this detection, DNA sample is incubated with a pair of capture sgRNAs (sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa and the signal readout-related probe is captured by the capture sequence of sgRNAb.Finally the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cell and virus. Especially, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples were successfully detected by the method. All detections can be finished in 30 minutes at room temperature. This detection holds promise for rapid on-the-spot detection or point-of-care testing (POCT).

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Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains

Cited by 144 publications

References 23 publications

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

CRISPR-Cas Systems for Diagnosing Infectious Diseases

CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique

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