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.

Cell-free protein synthesis (CFPS) has emerged as a novel protein expression platform. Especially the incorporation of non-canonical amino acids (ncAAs) has led to the development of numerous flexible methods for efficient and extensive expression of artificial proteins. Approaches were developed to eliminate the endogenous competition for ncAAs and engineer translation factors, which significantly enhanced the incorporation efficiency. Furthermore, in vitro aminoacylation methods can be conveniently combined with cell-free systems, extensively expanding the available ncAAs with novel and unique moieties. In this review, we summarize the recent progresses on the efficient and extensive incorporation of ncAAs by different strategies based on the elimination of competition by endogenous factors, translation factors engineering and extensive incorporation of novel ncAAs coupled with in vitro aminoacylation methods in CFPS. We also aim to offer new ideas to researchers working on ncAA incorporation techniques in CFPS and applications in various emerging fields.

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.

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.

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orthogonal suppressor tRNA holds the key to the incorporation efficiency. Recently, improved incorporation is achieved in E. coli strain C321.ΔA, in which all amber codons, accounting for 8% of stop codons in E. coli, [1] are replaced and prfA is removed away from the genome. [2] However, it will be extremely lethal for a cell to simultaneously interfere with all three stop codons. Saturation mutagenesis for molecular evolution is another application restricted by termination mechanisms. Stop codon involved in NNN randomized codon causes undesired premature termination. One stop codon is still generated even with NNK or NNS, moreover, the reduced codon set excludes codons with high usage frequency (Figure S1, Supporting Information). We anticipate that complete codon-dependent termination defect protein translation could efficiently liberate all codons for sense function and improve all the genetic code engineering applications (Figure 1).In the cell, termination of translation includes both the essential mechanism of class-I release factors and alternative mechanisms, [3] such as tmRNA (ssrA) mediated transtranslation [4] and translation stalling rescue. [5] As alternative termination ArfT in F. tularensis, [6] ResQ in Bacillus subtilis [7] are reported. In contrast to RF1, there is still no feasible approach for deletion of RF2, major class-I release factor in charge of the essential termination and plays a critical role in alternative terminations as well, i.e., post-peptidyl transfer quality control and alternative ribosome rescue. [8] However, cell viability becomes a barrier for in vivo assessment of global termination function. Therefore, there is strong curiosity about what if losing all essential termination machineries.Essential genes cannot be genetically deleted in a living cell. Pdt peptide containing 27 amino acids in length evolved from Mesoplasma florum Lon protease (mf-Lon) is demonstrated as being able to specifically target protein to efficient degradation in E. coli. [9] Pdt-tag is fused to nascent chain release factor RF1 and RRF respectively in E. coli MG1655pro, but failed on prfB encoding release factor RF2. Thus far, complete removal of all termination functions in either living cell or cell lysate was not yet achieved.Here, we present one efficient in vitro protein synthesis with 64 sense codons (iPSSC). The mf-Lon directed protein degradation is demonstrated as one efficient approach for the Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs...

Large scale DNA oligo pools are emerging as a novel material in a variety of advanced applications.However, GC content and length cause signi cant bias in ampli cation of oligos. We systematically explored the ampli cation of one oligo pool comprising of over ten thousand distinct strands with moderate GC content in the range of 35-65%. Uniqual ampli cation of oligos result to the increased Gini index of the oligo distribution while a few oligos greatly increased their proportion after 60 cycles of PCR. However, the signi cantly enriched oligos all have relatively high GC content. Further thermodynamic analysis demonstrated that a high value of both GC content and Gibbs free energy could improve the replication of speci c oligos during biased ampli cation. Therefore, this double-G (GC content and Gibbs free energy) driven replication advantage can be used as a guiding principle for the sequence design for a variety of applications, particularly for data storage.

45DNA emerged as novel material for mass data storage, the serious problem human society is facing. 46 Taking advantage of current synthesis capacity, massive oligo pool demonstrated its high-potential in 47 data storage in test tube. Herein, mixed culture of bacterial cells carrying mass oligo pool that was 48 assembled in a high copy plasmid was presented as a stable material for large scale data storage. Living 49 cells data storage was fabricated by a multiple-steps process, assembly, transformation and mixed culture. 50 The underlying principle was explored by deep bioinformatic analysis. Although homology assembly 51 showed sequence context dependent bias but the massive digital information oligos in mixed culture 52 were constant over multiple successive passaging. In pushing the limitation, over ten thousand distinct 53 oligos, totally 2304 Kbps encoding 445 KB digital data including texts and images, were stored in 54 bacterial cell, the largest archival data storage in living cell reported so far. The mixed culture of living 55 cell data storage opens up a new approach to simply bridge the in vitro and in vivo storage system with 56 combined advantage of both storage capability and economical information propagation. 57 1. Introduction: 58 While being biological material carrying genomic information, DNA has been proven of great potential 59 in storing information in its nucleic acid sequence for long-term in high density. The increased capability 60 of high throughput chip synthesis based writing and next generation sequencing based reading 61 technologies greatly advanced the development of synthesis nucleic acid mediated archival storage. 62Simply put, information was synthesized into DNA oligo molecule and then read out by sequencing. Till 63 now, a number of systems have been developed storing massive archival data into synthetic oligo pool. [1, 64 2] Classical electrical communication and computing algorithms such as Fountain and Reed-Solomon 65 code have been adapted for conversation of digital binary information to four letters nucleic acids 66 sequence and error correction. [3, 4] Restricted by current high throughput oligo synthesis techniques, oligo 67 with from 100 to around 200 nts in length was the major materials for information storage in test tube. 68However, the oligo size well fits with the major commercial sequencing platform, such as Illumina, [5] by 69 which sequence from 50 to 200 nucleotides can be obtained at single read from one oligo terminal end. 70Furthermore, the cost of chip-based synthesis achieved the lowest DNA synthesis, at least one or two 71 orders of magnitude lower than traditional column-based oligo synthesis. Thus far, as medium materials 72 synthesis oligo pool based in vitro system has been largely expanded for up to 200 MB information 73 storage. [6] 74 Besides test tubes, microbe cells are able to carry the synthesis DNA material with many advanced 75 features for archival information storage. In comparing with the cell-free in vitro syste...