Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage

Sun, Ruirui; Zhao, Yuqian; Wang, Wenjuan; Liu, Jun Jie; Chen, Chunlai

doi:10.1039/d2sc05463a

Cited by 14 publications

(17 citation statements)

References 75 publications

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“…Therefore, the update or assembly of Cas protein is also an efficient way to optimize the detection system. The update of the Cas protein may be achieved by rational design 122 or directed evolution. 123 Besides changing the protein, integrating Cas proteins of different types could improve the detection efficiency, too.…”

Section: ■ Future Outlookmentioning

confidence: 99%

“…For instance, recently, Sun et al revealed that a highly conserved positive-charge-enriched α helix in the Cas12a protein played a significant role in target searching and cleavage efficiency, and the target specificity can be modulated by engineering its residues, too. Through finding a key domain in Cas12a for target searching, this discovery pointed out a new strategy for Cas protein engineering, which may improve its specificity by a large margin . Studies on the mechanism could help us design the biosensor more efficiently and rationally, and more studies should be made on this aspect.…”

Section: Future Outlookmentioning

confidence: 99%

“…Through finding a key domain in Cas12a for target searching, this discovery pointed out a new strategy for Cas protein engineering, which may improve its specificity by a large margin. 122 Studies on the mechanism could help us design the biosensor more efficiently and rationally, and more studies should be made on this aspect.…”

Section: ■ Future Outlookmentioning

confidence: 99%

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Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

Li,

Cheng

2023

ACS Sens.

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Point-of-care (POC) detection is getting more and more attention in many fields due to its accuracy and on-site test property. The CRISPR/Cas12a system is endowed with excellent sensitivity, target identification specificity, and signal amplification ability in biosensing because of its unique trans-cleavage ability. As a result, a lot of research has been made to develop CRISPR/Cas12a-based biosensors. In this review, we focused on signal readout strategies and summarized recent sensitivity-improving strategies in fluorescence, colorimetric, and electrochemical signaling. Then we introduced novel portability-improving strategies based on lateral flow assays (LFAs), microfluidic chips, simplified instruments, and one-pot design. In the end, we also provide our outlook for the future development of CRISPR/Cas12a biosensors.

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Section: ■ Future Outlookmentioning

confidence: 99%

Section: Future Outlookmentioning

confidence: 99%

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Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

Li,

Cheng

2023

ACS Sens.

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“…In addition, Cas12a can process precursor crRNA (pre-crRNA) into mature crRNA, 16 which will obtain multiple crRNAs in one transcript simultaneously, achieving labeling multiple loci of the same target or different targets. Moreover, previous reports have demonstrated that PAM is also necessary for CRISPR/Cas12a during the target searching process, 17,18 analogous to CRISPR/ Cas9. Thus, we envisioned that the CRISPR/Cas12a system may possess more advantages for engineering an RNA imaging platform in live cells.…”

Section: ■ Introductionmentioning

confidence: 99%

“…These results verified the fact that the introduction of PAMmer(ACTB) is helpful to search for ACTB mRNA, consistent with the previous report of Cas12a/crRNA for asymmetrically searching the target flanking the PAM site. 17 In addition to the initial PAM recognition, the ability of specific recognition of the CRISPR/Cas12a system for the target is strongly influenced by the complementarity between the target and the seed region in crRNA. 25 To study the effect of mismatching in the seed region on RNA recognition by our proposed system, we designed a series of artificial crRNA sequences bearing one, two, and three nucleotide mismatches (m1, m2, m3) close to the first five bases of PAM in the seed region using a commonly overexpressed HER2 mRNA in breast cancer as a model 30 (Figure S6A).…”

Section: ■ Introductionmentioning

confidence: 99%

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells

Jia,

Zhang,

et al. 2024

Anal. Chem.

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CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAMpresenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.

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Ligand‐Responsive Artificial Protein–Protein Communication for Field‐Deployable Cell‐Free Biosensing

Wang,

Liu,

Zhou

et al. 2024

Angewandte Chemie

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Natural protein‐protein communications, such as those between transcription factors (TFs) and RNA polymerases/ribosomes, underpin cell‐free biosensing systems operating on the transcription/translation (TXTL) paradigm. However, their deployment in field analysis is hampered by the delayed response (hour‐level) and the complex composition of in vitro TXTL systems. For this purpose, we present a de novo‐designed ligand‐responsive artificial protein‐protein communication (LIRAC) by redefining the connection between TFs and non‐interacting CRISPR/Cas enzymes. By rationally designing a chimeric DNA adaptor and precisely regulating its binding affinities to both proteins, LIRAC immediately transduces target‐induced TF allostery into rapid CRISPR/Cas enzyme activation within a homogenous system. Consequently, LIRAC obviates the need for RNA/protein biosynthesis inherent to conventional TXTL‐based cell‐free systems, substantially reducing reaction complexity and time (from hours to 10 minutes) with improved sensitivity and tunable dynamic range. Moreover, LIRAC exhibits excellent versatility and programmability for rapidly and sensitively detecting diverse contaminants, including antibiotics, heavy metal ions, and preservatives. It also enables the creation of a multi‐protein communication‐based tristate logic for the intelligent detection of multiple contaminants. Integrated with portable devices, LIRAC has been proven effective in the field analysis of environmental samples and personal care products, showcasing its potential for environmental and health monitoring.

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Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage

Abstract: Cas12a is one of the most commonly-used Cas proteins for genome editing and gene regulation. The first key step for Cas12a to fulfill its function is to search for its...

Cited by 14 publications

References 75 publications

Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells

Ligand‐Responsive Artificial Protein–Protein Communication for Field‐Deployable Cell‐Free Biosensing

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