Daisy-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 2016

Transcriptome analysis in whole blood reveals increased microbial diversity in schizophrenia, bioRxiv, 2016-06-08

AbstractThe role of the human microbiome in health and disease is increasingly appreciated. We studied the composition of microbial communities present in blood across 192 individuals, including healthy controls and patients with three disorders affecting the brain schizophrenia, amyotrophic lateral sclerosis and bipolar disorder. By using high quality unmapped RNA sequencing reads as candidate microbial reads, we performed profiling of microbial transcripts detected in whole blood. We were able to detect a wide range of bacterial and archaeal phyla in blood. Interestingly, we observed an increased microbial diversity in schizophrenia patients compared to the three other groups. We replicated this finding in an independent schizophrenia case-control cohort. This increased diversity is inversely correlated with estimated cell abundance of a subpopulation of CD8+ memory T cells in healthy controls, supporting a link between microbial products found in blood, immunity and schizophrenia.

biorxiv microbiology 0-100-users 2016

Massively parallel clonal analysis using CRISPRCas9 induced genetic scars, bioRxiv, 2016-06-02

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scartrace, a strategy for massively parallel clonal analysis based on Cas9 induced genetic scars in the zebrafish.

biorxiv systems-biology 0-100-users 2016

Rapidly 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 2016

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, bioRxiv, 2016-05-23

AbstractThe CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Caseffector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage.In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

biorxiv molecular-biology 0-100-users 2016

Continuous 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 2016