CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn’t cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3′ overhang dsDNA substrates at least 3 times faster than 5′ overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule.
The physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.
more » « less- PAR ID:
- 10161233
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract DNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.
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Abstract Motivation As nanopore technology reaches ever higher throughput and accuracy, it becomes an increasingly viable candidate for reading out DNA data storage. Nanopore sequencing offers considerable flexibility by allowing long reads, real-time signal analysis, and the ability to read both DNA and RNA. We need flexible and efficient designs that match nanopore’s capabilities, but relatively few designs have been explored and many have significant inefficiency in read density, error rate, or compute time. To address these problems, we designed a new single-read per-strand decoder that achieves low byte error rates, offers high throughput, scales to long reads, and works well for both DNA and RNA molecules. We achieve these results through a novel soft decoding algorithm that can be effectively parallelized on a GPU. Our faster decoder allows us to study a wider range of system designs.
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Abstract T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions.
