An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype, whereas individuals homozygous for two copies of the allele will display a mutant phenotype. We ...have developed a method called the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations. In Drosophila, we found that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome, thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.
Drosophila melanogaster is emerging as one of the most effective tools for analyzing the function of human disease genes, including those responsible for developmental and neurological disorders, ...cancer, cardiovascular disease, metabolic and storage diseases, and genes required for the function of the visual, auditory and immune systems. Flies have several experimental advantages, including their rapid life cycle and the large numbers of individuals that can be generated, which make them ideal for sophisticated genetic screens, and in future should aid the analysis of complex multigenic disorders. The general principles by which D. melanogaster can be used to understand human disease, together with several specific examples, are considered in this review.
BMP mophogens direct growth and fate
As shown in classic fate-mapping studies, tissues and organs arise from specific regions of the embryo. Work over the past few decades has identified molecular ...players directing this choreographed development. Bone morphogenetic proteins (BMPs) and their antagonists establish domains in developing embryos. Bier and De Robertis review historical events for key discoveries in this area. They go on to lay out the current understanding of how diffusible morphogens form gradients to subdivide germ layers into distinct territories and organize body axes, regulate growth, and maintain stem cell niches.
Science
, this issue
10.1126/science.aaa5838
BACKGROUND
Classic embryological studies showed that diffusible factors (morphogens) influence cell fate during dorsal-ventral (DV) axis patterning. Subsequently, mathematical analyses applied reaction-diffusion equations in a theoretical framework to model how stable gradients of morphogenetic factors might be created in developing cell fields, according to the laws of physical chemistry. This work suggested mechanisms by which such gradients form and are read in a threshold-dependent fashion to establish distinct cellular responses. As highlighted in this Review, these pioneering experimental and intellectual insights laid the groundwork for more recent studies that have elucidated the mechanisms by which morphogen gradients are generated and stabilized by molecular feedback circuits.
ADVANCES
The molecular players involved in early DV patterning uncovered over the past two decades constitute a highly conserved cohort of extracellular factors that regulate bone morphogenetic protein (BMP) signaling. A key insight was the identification of the homologous proteins Short gastrulation (Sog) and Chordin as BMP-binding proteins in
Drosophila
and
Xenopus
20 years ago. Since then, analysis of this patterning system has led to dramatic advances in our understanding of the molecular mechanisms regulating early DV axis specification. Elements of this pathway include secreted BMP ligands and BMP antagonists, as well as extracellular metalloproteinases that cleave and inactivate BMP antagonists. Identification of these and other accessory proteins provided strong support for the proposal that an inversion of the DV axis had occurred between arthropods and vertebrates. Analysis of how these components are deployed in an array of species with divergent developmental strategies has deepened our understanding of this ancestral DV patterning biochemical pathway. These comparative studies have shed light on the broader question of a how a conserved core pathway can be modified during evolution to accommodate different forms of embryogenesis while maintaining common output effector functions. In addition, advances in computational analysis have provided the necessary tools to analyze BMP-mediated signaling in quantitative terms and have provided important insights into how this patterning process is integrated with cell proliferation and tissue growth. One such insight is the identification of expanders (such as Pentagone and Sizzled), which are secreted molecules typically produced at the low end of a gradient that stabilize the ligand, scaling the gradient to the growth of tissues.
OUTLOOK
An important unanswered question is how morphogen gradients form and function reliably in the face of intrinsic signal-degrading processes to achieve consistent developmental patterning and growth. One testable hypothesis, based on the “wisdom of crowds” concept, that may shed light on this challenging problem is that several independent features of morphogen gradients can be read in parallel by cells and can also serve as inputs to an array of feedback modules that integrate instantaneous levels of signaling, perform time averaging of signals, and act locally to coordinate signaling between neighboring cells. A consensus-based estimate of the relative position of a cell may be reached by deploying multiple parallel feedback modules. In addition, it will be important to determine the roles of mechanisms, such as free or facilitated diffusion in the extracellular space; exosomes; and cytonemes in morphogen gradient function. Understanding the mechanisms by which morphogen-mediated patterning systems evolve to maintain key elements of overall body design while allowing for a marked diversity in the spatial deployment of various subsets of signaling components is another compelling challenge. Such studies should better illuminate the precise nature of highly constrained developmental processes and delineate more fluid features of the networks that permit remodeling of core components to meet the specialized selective needs of particular organisms. These future studies should refine and strengthen one of the best paradigms for understanding development.
Conserved BMP-mediated patterning of the DV axis.
Gradients of proteins in vertebrates (left: blue Chordin stain) and invertebrates (left: red/yellow Sog stain) initiate patterning along the DV axis. These gradients are then read to establish distinct zones of gene expression within the central nervous system (right:
dpp
, yellow;
msh
, red;
ind
, green;
vnd
, blue).
CREDITS: CHORDIN GRADIENT (LEFT) COLORED AND MODIFIED BY PHOTOSHOP FROM FIG. 3A OF J. L. PLOUHINEC
ET AL
.,
PROC. NATL. ACAD. SCI. U.S.A.
110
, 20372–20379 (2013); SOG GRADIENT (RIGHT) MODIFIED BY PHOTOSHOP FROM FIG. 1B′′ OF S. SRINIVASAN
ET AL
.,
DEV. CELL
2
, 91–101 (2002)
Bone morphogenetic proteins (BMPs) act in dose-dependent fashion to regulate cell fate choices in a myriad of developmental contexts. In early vertebrate and invertebrate embryos, BMPs and their antagonists establish epidermal versus central nervous system domains. In this highly conserved system, BMP antagonists mediate the neural-inductive activities proposed by Hans Spemann and Hilde Mangold nearly a century ago. BMPs distributed in gradients subsequently function as morphogens to subdivide the three germ layers into distinct territories and act to organize body axes, regulate growth, maintain stem cell niches, or signal inductively across germ layers. In this Review, we summarize the variety of mechanisms that contribute to generating reliable developmental responses to BMP gradients and other morphogen systems.
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR-Cas9-based gene-drive systems are highly efficient in ...laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.
Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of ...introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.
DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Factors influencing the choice of particular repair pathways, however remain poorly characterized. Here ...we develop an Integrated Classification Pipeline (ICP) to decompose and categorize CRISPR/Cas9 generated mutations on genomic target sites in complex multicellular insects. The ICP outputs graphic rank ordered classifications of mutant alleles to visualize discriminating DSB repair fingerprints generated from different target sites and alternative inheritance patterns of CRISPR components. We uncover highly reproducible lineage-specific mutation fingerprints in individual organisms and a developmental progression wherein Microhomology-Mediated End-Joining (MMEJ) or Insertion events predominate during early rapid mitotic cell cycles, switching to distinct subsets of Non-Homologous End-Joining (NHEJ) alleles, and then to Homology-Directed Repair (HDR)-based gene conversion. These repair signatures enable marker-free tracking of specific mutations in dynamic populations, including NHEJ and HDR events within the same samples, for in-depth analysis of diverse gene editing events.
CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with ...20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14-15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.
Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs ...Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.
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