Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

Piskunen, Petteri; Latham, Rosalind; West, Claudia; Castronovo, Matteo; Linko, Veikko

doi:10.1016/j.isci.2022.104389

Cited by 9 publications

(8 citation statements)

References 94 publications

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“…These imaging results affirm the previous DNA gel data that a strong binding interaction exists between Cas12a and λ DNA. Although the interaction of self‐assembled DNA/RNA nanostructures to other small molecules, cells, and proteins (including Cas protein [27] ) has been studied previously,[ 27 , 28 ] the origin of binding affinity between Cas12a and λ DNA requires further investigation. In this perspective, a modeling approach [29] could help identify the specific regions of λ DNA that show an affinity to Cas12a in future.…”

Section: Resultsmentioning

confidence: 99%

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A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double‐Stranded λ DNA Reporter

2022

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CRISPR-based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR-Cas12a-based sensing platform to detect ssDNA targets by sizing double-stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massive trans-nuclease activity of Cas12a toward λ DNA is due to the presence of singlestranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes the trans-cleavage activity and helps achieve subpicomolar detection sensitivity, � 100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters.

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Section: Resultsmentioning

confidence: 99%

“…These imaging results affirm the previous DNA gel data that a strong binding interaction exists between Cas12a and λ DNA. Although the interaction of self-assembled DNA/RNA nanostructures to other small molecules, cells, and proteins (including Cas protein [27] ) has been studied previously, [27,28] the origin of binding affinity…”

Section: Methodsmentioning

confidence: 99%

A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double‐Stranded λ DNA Reporter

2022

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“…Similar applications can be fully tailored using synthetic and programmable DNA nanotechnology components [102], as has very recently been hinted at [89]. DNA origami in particular has seen increasing use as programmable drug delivery [103,104] and gene editing vectors [105] in the past few years. As such, we strongly believe that, in conjunction with CJNs and swimmers, DNA nanostructures will see growing interest when engineering custom-designed micro-and nanocarriers for TDDSs and other biomedical and non-medical applications.…”

Section: Discussionmentioning

confidence: 96%

Nanoswimmers Based on Capped Janus Nanospheres

2022

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Nanoswimmers are synthetic nanoscale objects that convert the available surrounding free energy to a directed motion. For example, bacteria with various flagella types serve as textbook examples of the minuscule swimmers found in nature. Along these lines, a plethora of artificial hybrid and non-hybrid nanoswimmers have been introduced, and they could find many uses, e.g., for targeted drug delivery systems (TDDSs) and controlled drug treatments. Here, we discuss a certain class of nanoparticles, i.e., functional, capped Janus nanospheres that can be employed as nanoswimmers, their subclasses and properties, as well as their various implementations. A brief outlook is given on different fabrication and synthesis methods, as well as on the diverse compositions used to prepare nanoswimmers, with a focus on the particle types and materials suitable for biomedical applications. Several recent studies have shown remarkable success in achieving temporally and spatially controlled drug delivery in vitro using Janus-particle-based TDDSs. We believe that this review will serve as a concise introductory synopsis for the interested readers. Therefore, we hope that it will deepen the general understanding of nanoparticle behavior in biological matrices.

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“…Spatial regulation of molecular machines has been demonstrated to implement directional motors, [15] cargo‐sorting walkers, [16] maze‐solving navigators [1] and other computational devices [1–3, 16, 17] . In addition, previous studies have shown that CRISPR systems could be anchored on DNA origami for enhancing and directing the delivery of CRISPR/Cas [18] and single‐molecule analysis [19] . In this study, we develop DNA origami‐based CRISPR molecular machines with spatially resolved activities.…”

Section: Figurementioning

confidence: 99%

DNA Origami‐Based Single‐Molecule CRISPR Machines for Spatially Resolved Searching

Hao

Zhang

et al. 2022

Angewandte Chemie

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Repurposing the RNA‐guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR‐Cas9‐based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami‐based single‐molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially‐confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high‐throughput nucleic acid detection.

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Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

Cited by 9 publications

References 94 publications

A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double‐Stranded λ DNA Reporter

A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double‐Stranded λ DNA Reporter

Nanoswimmers Based on Capped Janus Nanospheres

DNA Origami‐Based Single‐Molecule CRISPR Machines for Spatially Resolved Searching

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