Single-Nucleotide-Resolution Computing and Memory in Living Cells.

The ability to process and store information in living cells is essential for developing next-generation therapeutics and studying biology in situ. However, existing strategies have limited recording capacity and are challenging to scale. To overcome these limitations, we developed DOMINO, a robust and scalable platform for encoding logic and memory in bacterial and eukaryotic cells. Using an efficient single-nucleotide-resolution Read-Write head for DNA manipulation, DOMINO converts the living cells' DNA into an addressable, readable, and writable medium for computation and storage. DOMINO operators enable analog and digital molecular recording for long-term monitoring of signaling dynamics and cellular events. Furthermore, multiple operators can be layered and interconnected to encode order-independent, sequential, and temporal logic, allowing recording and control over the combination, order, and timing of molecular events in cells. We envision that DOMINO will lay the foundation for building robust and sophisticated computation-and-memory gene circuits for numerous biotechnological and biomedical applications.

Randomized CRISPR-Cas Transcriptional Perturbation Screening Reveals Protective Genes against Alpha-Synuclein Toxicity.

The genome-wide perturbation of transcriptional networks with CRISPR-Cas technology has primarily involved systematic and targeted gene modulation. Here, we developed PRISM (Perturbing Regulatory Interactions by Synthetic Modulators), a screening platform that uses randomized CRISPR-Cas transcription factors (crisprTFs) to globally perturb transcriptional networks. By applying PRISM to a yeast model of Parkinson's disease (PD), we identified guide RNAs (gRNAs) that modulate transcriptional networks and protect cells from alpha-synuclein (αSyn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of αSyn toxicity reported previously, highlighting PRISM's ability to identify modulators of important phenotypes. Gene expression profiling revealed genes differentially modulated by this strong protective gRNA that rescued yeast from αSyn toxicity when overexpressed. Human homologs of top-ranked hits protected against αSyn-induced cell death in a human neuronal PD model. Thus, high-throughput and unbiased perturbation of transcriptional networks via randomized crisprTFs can reveal complex biological phenotypes and effective disease modulators.

High-throughput functional variant screens via in vivo production of single-stranded DNA.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

Efficient retroelement-mediated DNA writing in bacteria.

The ability to efficiently and dynamically change information stored in genomes would enable powerful strategies for studying cell biology and controlling cellular phenotypes. Current recombineering-mediated DNA writing platforms in bacteria are limited to specific laboratory conditions, often suffer from suboptimal editing efficiencies, and are not suitable for in situ applications. To overcome these limitations, we engineered a retroelement-mediated DNA writing system that enables efficient and precise editing of bacterial genomes without the requirement for target-specific elements or selection. We demonstrate that this DNA writing platform enables a broad range of applications, including efficient, scarless, and cis-element-independent editing of targeted microbial genomes within complex communities, the high-throughput mapping of spatial information and cellular interactions into DNA memory, and the continuous evolution of cellular traits.

Emerging applications for DNA writers and molecular recorders.

Natural life is encoded by evolvable, DNA-based memory. Recent advances in dynamic genome-engineering technologies, which we collectively refer to as in vivo DNA writing, have opened new avenues for investigating and engineering biology. This Review surveys these technological advances, outlines their prospects and emerging applications, and discusses the features and current limitations of these technologies for building various genetic circuits for processing and recording information in living cells.

Synthetic biology. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations.

Cellular memory is crucial to many natural biological processes and sophisticated synthetic biology applications. Existing cellular memories rely on epigenetic switches or recombinases, which are limited in scalability and recording capacity. In this work, we use the DNA of living cell populations as genomic "tape recorders" for the analog and distributed recording of long-term event histories. We describe a platform for generating single-stranded DNA (ssDNA) in vivo in response to arbitrary transcriptional signals. When coexpressed with a recombinase, these intracellularly expressed ssDNAs target specific genomic DNA addresses, resulting in precise mutations that accumulate in cell populations as a function of the magnitude and duration of the inputs. This platform could enable long-term cellular recorders for environmental and biomedical applications, biological state machines, and enhanced genome engineering strategies.

Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas.

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function. Here, we show that the CRISPR/Cas system of Streptococcus pyogenes can be programmed to direct both activation and repression to natural and artificial eukaryotic promoters through the simple engineering of guide RNAs with base-pairing complementarity to target DNA sites. We demonstrate that the activity of CRISPR-based transcription factors (crisprTFs) can be tuned by directing multiple crisprTFs to different positions in natural promoters and by arraying multiple crisprTF-binding sites in the context of synthetic promoters in yeast and human cells. Furthermore, externally controllable regulatory modules can be engineered by layering gRNAs with small molecule-responsive proteins. Additionally, single nucleotide substitutions within promoters are sufficient to render them orthogonal with respect to the same gRNA-guided crisprTF. We envision that CRISPR-based eukaryotic gene regulation will enable the facile construction of scalable synthetic gene circuits and open up new approaches for mapping natural gene networks and their effects on complex cellular phenotypes.

Beta-sheet capping: signals that initiate and terminate beta-sheet formation.

In the present work, we address the question of whether different amino acids have different beta-sheet initiating and terminating characteristics. Using a large scale analysis of parallel and antiparallel beta-sheets in a non-redundant dataset of proteins, we observed that most of the amino acids show significant under- or over-representation in at least one of the positions at the two ends of beta-sheets, which are denoted as N-cap and C-cap. In addition, based on statistical data and structural comparison, we found that certain amino acids, especially Asp, Asn, Gly and Pro have strong tendencies to block beta-sheet continuation. Hence, we can consider these residues as beta-sheet terminators. It was also proposed that the dipole moments in parallel beta-sheets, whose direction is from C-terminal (partially negative) to N-terminal (partially positive), are much stronger than has previously been suggested. In fact, enhancement of dipole moments in parallel beta-sheets is a result of the positioning of positively charged residues at N-cap and negatively charged residues at C-cap. This enhancement in dipole moment magnitude leads to strengthened dipolar interactions between parallel beta-sheets dipoles and other partners especially alpha-helices dipoles. The results provide an explanation for the antiparallel alignment of parallel beta-sheets with alpha-helices.