Oligo replication advantage driven by GC content and Gibbs free energy.

Large scale DNA oligo pools are emerging as a novel material in a variety of advanced applications. However, GC content and length cause significant bias in amplification of oligos. We systematically explored the amplification of one oligo pool comprising of over ten thousand distinct strands with moderate GC content in the range of 35-65%. Uniqual amplification 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 significantly 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 specific oligos during biased amplification. 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.

Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d.

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 single-nucleotide variation with high specificity and sensitivity, thereby providing newly qualified machinery in toolbox for efficient molecular diagnostics.

Sustainable DNA Data Storage on Cellulose Paper.

DNA is a promising material for high density and long-term archival data storage. In addition to algorithms for encoding digital information into DNA sequences, the DNA writing (chemical synthesis) and reading (DNA sequencing), the preservation of DNA mixtures with high sequence diversity is another critical issue for sustainable, long-term, and large-scale DNA data storage. Here, this work demonstrates a method for low-cost, convenient and sustainable DNA data storage on cellulose paper. A DNA pool comprising thousands of sequences, in which archival data are encoded, is conveniently stored on a cellulose paper with a calculated density as high as 15 TB per mm3 through electrostatic adsorption. This work demonstrates that these digitally encoded DNA pools can be stable for years on the cellulose paper after drying even when directly exposed to air. Furthermore, the reversible electrostatic adsorption enables repeated loading/retrieval of DNA on/off cellulose paper. Therefore, this sustainable DNA preservation on cellulose paper through the convenient electrostatic adsorption exhibits a great advantage in terms of storage capacity and cost that is crucial for practical systems to achieve large-scale and long-time data storage.

Efficient In Vitro Full-Sense-Codons Protein Synthesis.

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 are screened for efficient all stop-codons decoding to construct a codon-dependent termination defect in vitro protein synthesis with all 64 sense-codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell.

Accurate genotyping of fragmented DNA using a toehold assisted padlock probe.

Fragmented DNA from blood plasma, i.e., cell-free DNA, has received great interest as a noninvasive diagnostic biomarker for "point-of-care" testing or liquid biopsy. Here, we present a new approach for accurate genotyping of highly fragmented DNA. Based on toehold-mediated strand displacement, a toehold-assisted padlock probe and toehold blocker were designed and demonstrated with new controllability in significantly suppressing undesired cross-reaction, promoting target recycling and point mutation detection by tuning the thermodynamic properties. Furthermore, toehold-assisted padlock probe systems were elaborately designed for 14 different single-nucleotide variants (SNVs) and were demonstrated to be able to detect low concentration of variant alleles (0.1%). In addition, a target, spanning a narrow sequence window of 29 nucleotides on average is sufficient for the toehold-assisted padlock probe system, which is valuable for the analysis of highly fragmented DNA molecules from clinical samples. We further demonstrated that the toehold-assisted padlock probe, in combination with a unique asymmetric PCR technique, could detect more target SNVs at low allele fractions (1%) in highly fragmented cfDNA. This allows accurate genotyping and provides a new commercial approach for high-resolution analysis of genetic variation.

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free 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.

A mixed culture of bacterial cells enables an economic DNA storage on a large scale.

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.

Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli.

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.