Enabling large-scale genome editing by reducing DNA nicking, bioRxiv, 2019-03-15
AbstractTo extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.One Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.
biorxiv synthetic-biology 200-500-users 2019Programmable patterns in a DNA-based reaction-diffusion system, bioRxiv, 2019-02-21
AbstractBiology offers compelling proof that macroscopic “living materials” can emerge from reactions between diffusing biomolecules. Here, we show that molecular self-organization could be a similarly powerful approach for engineering functional synthetic materials. We introduce a programmable DNA-hydrogel that produces tunable patterns at the centimeter length scale. We generate these patterns by implementing chemical reaction networks through synthetic DNA complexes, embedding the complexes in hydrogel, and triggering with locally applied input DNA strands. We first demonstrate ring pattern formation around a circular input cavity and show that the ring width and intensity can be predictably tuned. Then, we create patterns of increasing complexity, including concentric rings and non-isotropic patterns. Finally, we show “destructive” and “constructive” interference patterns, by combining several ring-forming modules in the gel and triggering them from multiple sources. We further show that computer simulations based on the reaction-diffusion model can predict and inform the programming of target patterns.
biorxiv synthetic-biology 0-100-users 2019Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins, bioRxiv, 2018-12-03
Protein therapy has the potential to alleviate many neurological diseases; however, delivery mechanisms for the central nervous system (CNS) are limited, and intracellular delivery poses additional hurdles. To address these challenges, we harnessed the protist parasite Toxoplasma gondii, which can migrate into the CNS and secrete proteins into cells. Using a fusion protein approach, we engineered T. gondii to secrete therapeutic proteins for human neurological disorders. We tested two secretion systems, generated fusion proteins that localized to the secretory organelles of T. gondii and assessed their intracellular targeting in various mammalian cells including neurons. We show that T. gondii expressing GRA16 fused to the Rett syndrome protein MeCP2 deliver a fusion protein that mimics the endogenous MeCP2, binding heterochromatic DNA in neurons. This demonstrates the potential of T. gondii as a therapeutic protein vector, which could provide either transient or chronic, in situ synthesis and delivery of intracellular proteins to the CNS.
biorxiv synthetic-biology 500+-users 2018Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator, bioRxiv, 2018-11-17
AbstractThe dynamics of the bacterial population that comprises the gut microbiota plays key roles in overall mammalian health. However, a detailed understanding of bacterial growth within the gut is limited by the inherent complexity and inaccessibility of the gut environment. Here, we deploy an improved synthetic genetic oscillator to investigate dynamics of bacterial colonization and growth in the mammalian gut under both healthy and disease conditions. The synthetic oscillator, when introduced into both Escherichia coli and Salmonella Typhimurium maintains regular oscillations with a constant period in generations across growth conditions. We determine the phase of oscillation from individual bacteria using image analysis of resultant colonies and thereby infer the number of cell divisions elapsed. In doing so, we demonstrate robust functionality and controllability of the oscillator circuit’s activity during bacterial growth in vitro, in a simulated murine gut microfluidic environment, and in vivo within the mouse gut. We determine different dynamics of bacterial colonization and growth in the gut under normal and inflammatory conditions. Our results show that a precise genetic oscillator can function in a complex environment and reveal single cell behavior under diverse conditions where disease may create otherwise impossible-to-quantify variability in growth across the population.
biorxiv synthetic-biology 0-100-users 2018beditor A computational workflow for designing libraries of guide RNAs for CRISPR-mediated base editing, bioRxiv, 2018-09-26
ABSTRACTCRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, PAM recognition sequences and genomes of many species. Additionally, in order to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates, called beditor scores are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces non-confounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base-editing to create or remove thousands of clinically important human disease mutations.
biorxiv synthetic-biology 0-100-users 2018Enzymatic DNA synthesis for digital information storage, bioRxiv, 2018-06-16
AbstractDNA is an emerging storage medium for digital data but its adoption is hampered by limitations of phosphoramidite chemistry, which was developed for single-base accuracy required for biological functionality. Here, we establish a de novo enzymatic DNA synthesis strategy designed from the bottom-up for information storage. We harness a template-independent DNA polymerase for controlled synthesis of sequences with user-defined information content. We demonstrate retrieval of 144-bits, including addressing, from perfectly synthesized DNA strands using batch-processed Illumina and real-time Oxford Nanopore sequencing. We then develop a codec for data retrieval from populations of diverse but imperfectly synthesized DNA strands, each with a ~30% error tolerance. With this codec, we experimentally validate a kilobyte-scale design which stores 1 bit per nucleotide. Simulations of the codec support reliable and robust storage of information for large-scale systems. This work paves the way for alternative synthesis and sequencing strategies to advance information storage in DNA.
biorxiv synthetic-biology 100-200-users 2018