Resetting the yeast epigenome with human nucleosomes, bioRxiv, 2017-06-08
SummaryHumans and yeast are separated by a billion years of evolution, yet their conserved core histones retain central roles in gene regulation. Here, we “reset” yeast to use core human nucleosomes in lieu of their own, an exceedingly rare event which initially took twenty days. The cells adapt, however, by acquiring suppressor mutations in cell-division genes, or by acquiring certain aneuploidy states. Robust growth was also restored by converting five histone residues back to their yeast counterparts. We reveal that humanized nucleosomes in yeast are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy and reduce RNA content. Adaptation to new biological conditions presented a special challenge for these cells due to slower chromatin remodeling. This humanized yeast poses many fundamental new questions about the nature of chromatin and how it is linked to many cell processes, and provides a platform to study histone variants via yeast epigenome reprogramming.Highlights<jatslist list-type=simple><jatslist-item>- Only 1 in 107 yeast survive with fully human nucleosomes, but they rapidly evolve<jatslist-item><jatslist-item>- Nucleosome positioning and nucleosome repeat length is not influenced by histone type<jatslist-item><jatslist-item>- Human nucleosomes remodel slowly and delay yeast environmental adaptation<jatslist-item><jatslist-item>- Human core nucleosomes are more repressive and globally reduce transcription in yeast<jatslist-item>
biorxiv synthetic-biology 0-100-users 2017DNA Fountain enables a robust and efficient storage architecture, bioRxiv, 2016-09-10
AbstractDNA is an attractive medium to store digital information. Here, we report a storage strategy, called DNA Fountain, that is highly robust and approaches the information capacity per nucleotide. Using our approach, we stored a full computer operating system, movie, and other files with a total of 2.14 × 106 bytes in DNA oligos and perfectly retrieved the information from a sequencing coverage equivalent of a single tile of Illumina sequencing. We also tested a process that can allow 2.18 × 1015 retrievals using the original DNA sample and were able to perfectly decode the data. Finally, we explored the limit of our architecture in terms of bytes per molecules and obtained a perfect retrieval from a density of 215Petabytegram of DNA, orders of magnitudes higher than previous techniques.
biorxiv synthetic-biology 100-200-users 2016Daisy-chain gene drives for the alteration of local populations, bioRxiv, 2016-06-08
AbstractRNA-guided gene drive elements could address many ecological problems by altering the traits of wild organisms, but the likelihood of global spread tremendously complicates ethical development and use. Here we detail a localized form of CRISPR-based gene drive composed of genetic elements arranged in a daisy-chain such that each element drives the next. “Daisy drive” systems can duplicate any effect achievable using an equivalent global drive system, but their capacity to spread is limited by the successive loss of non-driving elements from the base of the chain. Releasing daisy drive organisms constituting a small fraction of the local wild population can drive a useful genetic element to local fixation for a wide range of fitness parameters without resulting in global spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionary stable daisy drive as well as global CRISPR-based gene drive. Daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.Author’s Summary‘Global’ gene drive systems based on CRISPR are likely to spread to every population of the target species, hampering safe and ethical use. ‘Daisy drive’ systems offer a way to alter the traits of only local populations in a temporary manner. Because they can exactly duplicate the activity of any global CRISPR-based drive at a local level, daisy drives may enable safe field trials and empower local communities to make decisions concerning their own shared environments.For more details and an animation intended for a general audience, see the summary at Sculpting Evolution.
biorxiv synthetic-biology 0-100-users 2016Rapidly evolving homing CRISPR barcodes, bioRxiv, 2016-05-28
AbstractWe present here an approach for engineering evolving DNA barcodes in living cells. The methodology entails using a homing guide RNA (hgRNA) scaffold that directs the Cas9-hgRNA complex to target the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cultured cell populations and show that they can record lineage history and and that their RNA can be assayed as single molecules in situ. This integrated approach will have wide ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.
biorxiv synthetic-biology 0-100-users 2016Continuous Genetic Recording with Self-Targeting CRISPR-Cas in Human Cells, bioRxiv, 2016-05-23
AbstractThe ability to longitudinally track and record molecular events in vivo would provide a unique opportunity to monitor signaling dynamics within cellular niches and to identify critical factors in orchestrating cellular behavior. We present a self-contained analog memory device that enables the recording of molecular stimuli in the form of DNA mutations in human cells. The memory unit consists of a self-targeting guide RNA (stgRNA) cassette that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity towards the DNA that encodes the stgRNA, thereby enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We analyze the temporal sequence evolution dynamics of stgRNAs containing 20, 30 and 40 nucleotide SDSes (Specificity Determining Sequences) and create a population-based recording metric that conveys information about the duration andor intensity of stgRNA activity. By expressing stgRNAs from engineered, inducible RNA polymerase (RNAP) III promoters, we demonstrate programmable and multiplexed memory storage in human cells triggered by doxycycline and isopropyl β-D-1-thiogalactopyranoside (IPTG). Finally, we show that memory units encoded in human cells implanted in mice are able to record lipopolysaccharide (LPS)-induced acute inflammation over time. This tool, which we call Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a unique strategy for investigating cell biology in vivo and in situ and may drive further applications that leverage continuous evolution of targeted DNA sequences in mammalian cells.One Sentence SummaryBy designing self-targeting guide RNAs that repeatedly direct Cas9 nuclease activity towards their own DNA, we created multiplexed analog memory operators that can record biologically relevant information in vitro and in vivo, such as the magnitude and duration of exposure to TNAα.
biorxiv synthetic-biology 0-100-users 2016RNA-guided gene drives can efficiently and reversibly bias inheritance in wild yeast, bioRxiv, 2015-01-17
Inheritance-biasing “gene drives” may be capable of spreading genomic alterations made in laboratory organisms through wild populations. We previously considered the potential for RNA-guided gene drives based on the versatile CRISPRCas9 genome editing system to serve as a general method of altering populations. Here we report molecularly contained gene drive constructs in the yeast Saccharomyces cerevisiae that are typically copied at rates above 99% when mated to wild yeast. We successfully targeted both non-essential and essential genes, showed that the inheritance of an unrelated “cargo” gene could be biased by an adjacent drive, and constructed a drive capable of overwriting and reversing changes made by a previous drive. Our results demonstrate that RNA-guided gene drives are capable of efficiently biasing inheritance when mated to wild-type organisms over successive generations.
biorxiv synthetic-biology 0-100-users 2015