CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus

Yoshimi, Kazuto; Takeshita, Keizo; Yamayoshi, Seiya; Shibumura, Satomi; Yamauchi, Yuko; Yamamoto, Masaki; Yotsuyanagi, Hiroshi; Kawaoka, Yoshihiro; Mashimo, Tomoji

doi:10.1016/j.isci.2022.103830

Cited by 44 publications

(30 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To quantitatively measure collateral ssDNA cleavage activity we used a FQ-labeled untargeted ssDNA probe 34 , 43 (Fig. 1c ), which is used in CRISPR-based diagnostics as a platform for rapid and sensitive nucleic acid detection, for example in Covid-19 test kits 45 – 47 . Consistent with the results of the M13/linear ssDNA cleavage (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To analyze DNA cleavage activity, 1.6 nM of plasmid with or without targeted sequences were added to 115 nM EcoCascade-crRNA complex, 250 nM EcoCas3, and 2.5 mM ATP in CRISPR-Cas3 working buffer (60 mM KCl, 10 mM MgCl 2 , 10 µM CoCl 2 , 5 mM HEPES-KOH, pH 7.5), as previously described 34 , 43 , 47 . After incubation at 37 °C, samples were detected by either electrophoresis or with the MultiNa microchip electrophoresis system and the DNA-12,000 kit (Shimadzu, Kyoto, Japan).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Yoshimi

Takeshita²,

Kodera

et al. 2022

Nat Commun

Self Cite

View full text Add to dashboard Cite

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific single-stranded DNA (ssDNA) cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target double-stranded DNA (dsDNA) substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Yoshimi

Takeshita²,

Kodera

et al. 2022

Nat Commun

Self Cite

View full text Add to dashboard Cite

show abstract

“…Diagnostic CRISPR systems have outstanding superiority in terms of their ultrahigh sensitivity, portability, and specificity. For instance, the Cas12 and Cas13 nucleases have been reprogrammed to detect the nucleic acids of SARS‐CoV‐2 134–136 …”

Section: Genome Editing For Fundamental Research: Disease Modelling A...mentioning

confidence: 99%

“…For instance, the Cas12 and Cas13 nucleases have been reprogrammed to detect the nucleic acids of SARS-CoV-2. [134][135][136] Viruses, whether DNA viruses or RNA viruses, are important pathogens of infectious diseases. RNA viruses that have been studied abundantly in recent years include dengue, 137 Zika, 49 and a recent spotlight of interest, SARS-CoV-2.…”

Section: New Disease Diagnostic Tools Based On the Crispr-cas Systemmentioning

confidence: 99%

Current landscape of gene‐editing technology in biomedicine: Applications, advantages, challenges, and perspectives

Zhou

Yang

Zhang

et al. 2022

MedComm

View full text Add to dashboard Cite

The expanding genome editing toolbox has revolutionized life science research ranging from the bench to the bedside. These “molecular scissors” have offered us unprecedented abilities to manipulate nucleic acid sequences precisely in living cells from diverse species. Continued advances in genome editing exponentially broaden our knowledge of human genetics, epigenetics, molecular biology, and pathology. Currently, gene editing‐mediated therapies have led to impressive responses in patients with hematological diseases, including sickle cell disease and thalassemia. With the discovery of more efficient, precise and sophisticated gene‐editing tools, more therapeutic gene‐editing approaches will enter the clinic to treat various diseases, such as acquired immunodeficiency sydrome (AIDS), hematologic malignancies, and even severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. These initial successes have spurred the further innovation and development of gene‐editing technology. In this review, we will introduce the architecture and mechanism of the current gene‐editing tools, including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated nuclease‐based tools and other protein‐based DNA targeting systems, and we summarize the meaningful applications of diverse technologies in preclinical studies, focusing on the establishment of disease models and diagnostic techniques. Finally, we provide a comprehensive overview of clinical information using gene‐editing therapeutics for treating various human diseases and emphasize the opportunities and challenges.

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus

Cited by 44 publications

References 61 publications

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Current landscape of gene‐editing technology in biomedicine: Applications, advantages, challenges, and perspectives

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

Contact Info

Product

Resources

About