Nano for CRISPR

Hu, Tony; Chen, Xiaodong

doi:10.1021/acsnano.2c05431

Cited by 7 publications

(9 citation statements)

References 10 publications

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“…Nanomaterial-based approaches are well suited to enable intracellular sensing. 1 Numerous strategies involving nanoparticles, nanorods, and nanowires have been extensively explored. 20,21 In particular, nanoneedles, vertically aligned arrays of high aspect ratio nanostructures, have been widely used to access the intracellular space with minimal disruption of cell functions.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) (CRISPR/Cas) system, originally identified in bacteria and archaea, has gained considerable attention in molecular diagnostics due to its superb specificity of molecular target recognition, fast turnaround time, convenient isothermal reaction, and signal amplification capabilities. – Several CRISPR-based sensing approaches such as DETECTR (DNA endonuclease-targeted CRISPR trans reporter), SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), HOLMES (1 h low-cost multipurpose highly efficient system), and others, have been developed with high sensitivity and specificity. – For example, DETECTR showed attomolar sensitivity; HOLMES achieved a limit of detection (LOD) around 10 aM, and SHERLOCKv2 enabled the multiplexed sensing of target detection at zeptomolar sensitivity. – Among diverse Cas proteins, Cas12a (a class of type V nuclease) shows a unique collateral cleavage activity that can nonspecifically cleave single-stranded DNA substrates (termed as trans -cleavage) upon recognition of the correct target DNA by crRNA. , By adopting single-stranded DNA labeled with a fluorophore and quencher pair, the collateral cleavage activity can generate enormously amplified signals in response to a target analyte, including not only nucleic acids but also proteins, transcription factors, and small molecules. – In particular, Cas12a-based sensors can rapidly and sensitively detect adenosine triphosphate (ATP) levels. – Yet Cas12a collateral cleavage is not suited to in situ intracellular detection as it requires the codelivery of multiple components (the Cas12a RNP, the crRNA, and the DNA substrates) and risks indiscriminate off-target cleavage of nucleic acids essential to cell function. , Such a limitation prevents the use of this sensitive and specific sensing approach to monitor live cells.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells

Kim,

Gu,

Mustfa

et al. 2023

ACS Appl. Mater. Interfaces

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The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells

Kim,

Gu,

Mustfa

et al. 2023

ACS Appl. Mater. Interfaces

View full text Add to dashboard Cite

show abstract

“…Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)associated nuclease (Cas) (CRISPR/Cas) system, originally identified in bacteria and archaea, has gained considerable attention in molecular diagnostics due to its superb specificity of molecular target recognition, fast turnaround time, convenient isothermal reaction, and signal amplification capabilities. [1][2][3] Several CRISPR-based sensing approaches such as DETECTR (DNA endonuclease-targeted CRISPR trans reporter), SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), HOLMES (one-hour low-cost multipurpose highly efficient system), and others, have been developed with high sensitivity and specificity. [4][5][6][7][8][9] For example, DETECTR showed attomolar sensitivity, HOLMES achieved a limit of detection (LOD) around 10 aM, and SHERLOCKv2 enabled the multiplexed sensing of target detection at zeptomolar sensitivity.…”

Section: Introductionmentioning

confidence: 99%

“…Nanomaterial-based approaches are well suited to enable intracellular sensing. 3 Numerous strategies involving nanoparticles, nanorods, and nanowires have been extensively explored. 20,21 In particular, nanoneedles, vertically-aligned arrays of high aspect ratio nanostructures, 22 have been widely used to access the intracellular space with minimal disruption of cell functions and sense intracellular biomolecules.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas-assisted Nanoneedle Sensor for ATP Detection in Living Cells

Kim

Mustfa

et al. 2022

Preprint

View full text Add to dashboard Cite

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as a powerful molecular biosensing tool based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of collateral cleavage activity for in-situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of a ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 μM to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broaden the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.

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“…At minimum such studies should use disease-positive and -negative samples derived from the same population and collected under the same protocol to avoid data artifacts that may occur when using unrelated sample cohorts. In the conclusion and discussion section, studies must put into context how their findings advance translation and discuss hurdles and challenges anticipated moving forward. − …”

mentioning

confidence: 99%