CRISPR-based DNA and RNA detection with liquid-liquid phase separation

Spoelstra, Willem Kasper; Jacques, Jeroen M.; Gonzalez-Linares, Rodrigo; Nóbrega, Franklin L.; Haagsma, Anna C.; Dogterom, Marileen; Meijer, Dimphna H.; Idema, Timon; Brouns, Stan J. J.; Reese, Louis

doi:10.1016/j.bpj.2021.02.013

Cited by 25 publications

(17 citation statements)

References 49 publications

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“…Cas12a collateral activity was pivotal in efforts to simplify the diagnostic approach. Without compromising sensitivity or specificity, CRISPR-Cas12a provided a visual interpretation of the results via a turbidity, blue light, or lateral flow analysis (LFA) ( Figure 5 ) [ 32 , 60 , 79 ]. To prevent aerosol contamination during the uncapping of the reaction tube, the visual Cas12a-based platform “Cas12a-VDet” was devised.…”

Section: Crispr/cas Tools For Nucleic Acid Detectionmentioning

confidence: 99%

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Bhardwaj

Kant

Behera

et al. 2022

IJMS

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The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.

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Section: Crispr/cas Tools For Nucleic Acid Detectionmentioning

confidence: 99%

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Bhardwaj

Kant

Behera

et al. 2022

IJMS

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“…On target recognition, collateral cleavage results in the breakdown of nucleic acid polymers. After complementation with polycations, the solution either remains clear when cleavage has happened or becomes turbid when no target-triggered cleavage has occurred [87]. In another study, Cas13a collaterally cleaves the uracil ribonucleotide (rU)-bearing pre-primer upon recognition of target RNA.…”

Section: Readout Optionsmentioning

confidence: 99%

CRISPR Approaches for the Diagnosis of Human Diseases

Puig-Serra

Casado-Rosas

Martínez-Lage

et al. 2022

IJMS

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CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.

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“…Further options combine the amplification and detection stages with automation (Ackerman et al, 2020;Arizti-Sanz et al, 2020;Kellner et al, 2019;Qin et al, 2019). To apply SHERLOCK in harsh field conditions, different readout techniques have been proposed that include lateral flow paper strips (Arizti-Sanz et al, 2020;Barnes et al, 2020;Gootenberg et al, 2018;Myhrvold et al, 2018), and estimation of solution turbidity changes by eye through liquidliquid phase separation (Spoelstra et al, 2021). An interesting idea to aid in the interpretation of low-concentration readouts is the use of a mobile phone application.…”

Section: Detection Of Sars-cov-2 and Other Virusesmentioning

confidence: 99%

Applications of the versatile CRISPR‐Cas13 RNA targeting system

Kordyś

Sen²,

Warkocki³

2021

WIREs RNA

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CRISPR-Cas are adaptable natural prokaryotic defense systems that act against invading viruses and plasmids. Among the six currently known major CRISPR-Cas types, the type VI CRISPR-Cas13 is the only one known to exclusively bind and cleave foreign RNA. Within the last couple of years, this system has been adapted to serve numerous, and sometimes not obvious, applications, including some that might be developed as effective molecular therapies. Indeed, Cas13 has been adapted to kill antibiotic-resistant bacteria. In a cellfree environment, Cas13 has been used in the development of highly specific, sensitive, multiplexing-capable, and field-adaptable detection tools. Importantly, Cas13 can be reprogrammed and applied to eukaryotes to either combat pathogenic RNA viruses or in the regulation of gene expression, facilitating the knockdown of mRNA, circular RNA, and noncoding RNA. Furthermore, Cas13 has been harnessed for in vivo RNA modifications including programmable regulation of alternative splicing, A-to-I and C to U editing, and m6A modifications. Finally, approaches allowing for the detection and characterization of RNA-interacting proteins have also been demonstrated. Here, we provide a comprehensive overview of the applications utilizing CRISPR-Cas13 that illustrate its versatility. We also discuss the most important limitations of the CRISPR-Cas13-based technologies, and controversies regarding them. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications K E Y W O R D S bactericidal agents, CRISPR-Cas13, gene knockdown, RNA editing, RNA methods | INTRODUCTIONOver the last three decades, several seminal discoveries not only advanced general knowledge of RNA and RNA metabolism in living organisms and viruses, but also they led to the development of RNA-targeted molecular tools. These have opened up new opportunities for scientific and therapeutic applications. The big breakthrough at the turn of this

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CRISPR-based DNA and RNA detection with liquid-liquid phase separation

Cited by 25 publications

References 49 publications

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

CRISPR Approaches for the Diagnosis of Human Diseases

Applications of the versatile CRISPR‐Cas13 RNA targeting system

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