CRISPR-Cas12a-assisted nucleic acid detection

Li, Shi‐Yuan; Cheng, Qiuxiang; Wang, Jing-Man; Li, Xiaoyan; Zhang, Zilong; Gao, Song; Cao, Ruibing; Zhao, Guoping; Wang, Jin

doi:10.1038/s41421-018-0028-z

Cited by 1,019 publications

(813 citation statements)

References 12 publications

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“…The studies of the thermophilic Agos have revealed unique mechanistic features but still remain elusive in the feasibility of new gDNA in the canonical DNA cleavage reaction during the catalytic process. Of particular note biotechnologically, the nucleic acid-guided DNase activity of the CRISPR-associated protein has revolutionized the genome-detecting field recently [13][14][15][16], thus the thermophilic Agos were postulated suitable candidates for DNA reprograming applications [17,18]. Here, we sought to address the mentioned question using in vitro assays with PfAgo testing various gDNAs and substrates.…”

Section: Main Textmentioning

confidence: 99%

The stepwise endonuclease activity of a thermophilic Argonaute protein

Xun

Liu

Chong

et al. 2019

Preprint

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Thermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5'-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the PfAgo stepwise endonuclease activity followed by target DNA amplification, we establish a rapid and specific platform for the unambiguously multiplex gene detection, termed RADAR (Renewed-gDNA Assisted DNA-cleavage by Argonaute). In the end, RADAR was applied to distinguish of human papillomavirus of serotypes in patient samples in a single reaction, suggesting that our technique would be adopted for diagnosing application.

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Section: Main Textmentioning

confidence: 99%

The stepwise endonuclease activity of a thermophilic Argonaute protein

Xun

Liu

Chong

et al. 2019

Preprint

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“…Loop-Mediated Isothermal Amplification (LAMP) (12)), the target concentration 15 could decrease to 10 -8 nM ( Fig. 2a), which was the same as that of Cas12a (7). Therefore, because of the high sensitivity for DNA substrate, Cas12b can also be employed for nucleic acid detection, and the new method combining both amplification and Cas12b trans-cleavage detection is named HOLMESv2.…”

Section: Main Textmentioning

confidence: 96%

“…Due to the high sensitivity, specificity and simplicity, the recently emerged CRISPR-based nucleic acid detection methods have shown great promise in clinical diagnostics and have therefore drawn much attention (1,2). The principle of these methods is based on the trans-cleavage activities of some Cas proteins, including the DNA-targeting and single-stranded DNA (ssDNA)-cleaving 5 Cas12a (3)(4)(5)(6)(7)(8). Combined with amplification, Cas12a has been employed to develop HOLMES (one-HOur Low-cost Multipurpose highly Efficient System) or DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) (3,6,7).…”

Section: Main Textmentioning

confidence: 99%

“…Besides of DNA, RNA can also be detected with the CRISPR-based methods; however, RNA needs to be first reverse transcribed into cDNA, which is then used as the template for either in vitro transcription (e.g. SHERLOCK or SHERLOCKv2) or amplification (HOLMES or DETECTR) (3)(4)(5)(6)(7)(8). Although reverse transcription and subsequent amplification can be integrated into a one-pot system, the whole process contains more than one enzyme and can be complicated.…”

Section: Main Textmentioning

confidence: 99%

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CRISPR-Cas12b-assisted nucleic acid detection platform

Li²,

Wang

2018

Preprint

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Rapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-5 Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step. 10 One Sentence Summary: We combine LAMP and Cas12b to develop HOLMESv2 for conveniently detecting target nucleic acid in a one-step approach.

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“…In the recent past, studies, employing clustered regularly interspaced short palindromic repeats (CRISPR) associated methods for the detection of nucleic acids, have been utilized using different Cas effectors 14 . Varying Cas effectors were used for targeting different nucleic acids, like the Cas9 effector for the detection of dsDNA [15][16][17] , along with the Cas12 for the detection of ssDNA [18][19][20][21] . The microbial CRISPR effector Cas13a (previously named C2c2), displays, in contrast to the other Cas effectors, a triggered cleavage capability of non-target ssRNAs in the surrounding 22,23 .…”

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confidence: 99%

CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

Bruch

Baaske

Chatelle

et al. 2019

Preprint

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Non-coding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated to many different diseases, such as cancer, dementia or cardiovascular conditions. The key for an effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. Here, we introduce a CRISPR/Cas13a powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a has been realized without any nucleic acid amplification. With a readout time of 9 minutes and an overall process time of less than 4 hours, a limit of detection of 10 pM was achieved, using a measuring volume of less than 0.6 µl. Furthermore, we demonstrate the feasibility of our versatile sensor platform to detect miR-19b in serum samples of children, suffering from brain cancer. The validation of our results with a standard qRT-PCR method shows the ability of our system to be a low-cost and target amplification-free tool for nucleic acid based diagnostics.MicroRNAs (miRNAs) are composed of 18-25 nucleotides in their mature form and play an essential role as regulators of gene expression in many biological processes by binding to specific messenger RNA targets and promoting their degradation or translational inhibition 1,2 . In recent years, the popularity of miRNAs in research has been constantly growing, as the presence or dysregulation of distinct miRNAs can indicate specific medical conditions. For instance, in cancer research the up or down regulation of miRNA expression levels has been linked to certain cancer types, including lung cancer or brain tumors. This offers new possibilities for the detection and monitoring of such diseases, which is why miRNAs will continue to come in the focus of clinical diagnostics 3-5 .

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CRISPR-Cas12a-assisted nucleic acid detection

Cited by 1,019 publications

References 12 publications

The stepwise endonuclease activity of a thermophilic Argonaute protein

The stepwise endonuclease activity of a thermophilic Argonaute protein

CRISPR-Cas12b-assisted nucleic acid detection platform

CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

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