DNA emerged as a novel potential material for mass data storage, offering the possibility to cheaply solve a great data storage problem. Large oligonucleotide pools demonstrated high potential of large-scale data storage in test tube, meanwhile, living cell with high fidelity in information replication. Here we show a mixed culture of bacterial cells carrying a large oligo pool that was assembled in a high-copy-number plasmid was presented as a stable material for large-scale data storage. The underlying principle was explored by deep bioinformatic analysis. Although homology assembly showed sequence context dependent bias, the large oligonucleotide pools in the mixed culture were constant over multiple successive passages. Finally, over ten thousand distinct oligos encompassing 2304 Kbps encoding 445 KB digital data, were stored in cells, the largest storage in living cells reported so far and present a previously unreported approach for bridging the gap between in vitro and in vivo systems.

In DNA data storage, the massive sequence complexity creates challenges in repeatable and efficient information readout. Here, our study clearly demonstrated that PCR created significant DNA amplification biases due to its inherent mechanism of inefficient priming, product-as-template, and error-spreading prone, which greatly hinder subsequent applications such as data retrieval in DNA-based storage. To mitigate the amplification bias, we recruited an isothermal DNA amplification by combining strand displacement amplification (SDA) with magnetic beads (MB) DNA immobilization for robust, repeated, and low-bias amplification of DNA oligo pool, comprising over 100 thousand oligos, in a primer-free and low-error-spreading fashion. Furthermore, we introduced oligo pool normalization (OPN), a cost-effective and scalable method for normalizing an oligo pool, by which oligo pools comprising from 256 to 1024 distinct oligos were simply modified with improved Gini-index. Therefore, we believe that the combination of SDA and OPN can provide an ideal amplification mechanism for a low-bias copy of a large oligo pool, which is of vital importance for successful data retrieval in DNA information storage.

RNA-guided CRISPR (RNA-targeting clustered regularly interspaced short palindromic repeats) effector Cas13d is the smallest Class II subtype VI proteins identified so far. Here, two recently identified Cas13d effectors from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) were characterized and applied for sensitive nucleic acid detection. We demonstrated that the special target triggered collateral cleavage of these two Cas13d orthologs could provide rapid target RNA detection in picomolar range and then the tolerance for mismatch between crRNA and target RNA was characterized as well. Finally, an additional single mismatch was introduced into crRNA to enhance the two Cas13d orthologs mediated detection of low variant allele fraction, 0.1% T790M. Overall, this study demonstrated that both EsCas13d and RspCas13d could robustly detect target RNA carrying special singlenucleotide variation with high specificity and sensitivity, thereby providing newly qualified machinery in toolbox for efficient molecular diagnostics.

No abstract

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.