Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9–sgRNA Ribonucleoproteins

We present a novel method of targeted gene disruption that involves direct injection of recombinant Cas9 protein complexed with guide RNA into the gonad of the nematode Caenorhabditis elegans. Biallelic mutants were recovered among the F₁ progeny, demonstrating the high efficiency of this method.     

Somatic CRISPR–Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2–driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT–dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.     

CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.     

Mechanosensitive body–brain interactions in Caenorhabditis elegans

Proprioception and visceral mechanosensation provide important information about the location and deformation of the body parts in space and time. These deformations arise from muscle contraction during locomotion, but also from volume changes in organs that are subjected to stresses as a part of their physiological function. These internal morphodynamics give rise to periodic contraction–relaxation cycles with surprisingly constant amplitudes and the maintenance of these optimal driving patterns is remarkably robust against external and internal perturbations. One of the underlying reason for this robustness is an internal feedback mechanism in which specialized sensory cells and neurons signal the mechanical deformation of the inner workings of our organs, from the body to the brain, which subsequently adjust the driver to a predetermined physiological setpoint. Here, we review recent progress in the field of visceral mechanosensation and proprioception in Caenorhabditis elegans and discuss how future studies with this model can be used to gain insight into mechanosensory body–brain interactions in mammals.     

Knockouts of a late flowering gene via CRISPR–Cas9 confer early maturity in rice at multiple field locations

Plant Molecular Biology - OsGhd7 gene was discovered by screening our rice activation tagging population. CRISPR–Cas9 created knockouts of OsGhd7 conferred early flowering and early maturity...     

A Putative Catenin–Cadherin System Mediates Morphogenesis of the Caenorhabditis elegans Embryo

During morphogenesis of the Caenorhabditis elegans embryo, hypodermal (or epidermal) cells migrate to enclose the embryo in an epithelium and, subsequently, change shape coordinately to elongate the body (Priess, J.R., and D.I. Hirsh. 1986. Dev. Biol. 117:156– 173; Williams-Masson, E.M., A.N. Malik, and J. Hardin. 1997. Development [Camb.]. 124:2889–2901). We have isolated mutants defective in morphogenesis that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins α-catenin, β-catenin/Armadillo, and classical cadherin, respectively. This putative catenin–cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization.The morphology of the animal body and its tissues arise as embryonic cells change their shapes and/or positions (Mittenthal and Jacobson, 1990). Many of these changes are mediated by dynamic rearrangements of cytoskeletal components (Wessells et al., 1971). Cells can organize diverse patterns of microtubules and actin filaments, and movement of actin filaments by myosin proteins is thought to generate the force that drives many morphogenetic processes. An important step toward understanding the mechanical basis of morphogenesis is the identification and characterization of molecules that pattern the cytoskeleton and translate force into concerted cell movements. For cells to change shape coordinately or move relative to each other, forces generated within an individual cell must be transmitted to adhesive junctions at the plasma membrane and exerted on neighboring cells or the extracellular matrix (Gumbiner, 1996). The best characterized cell–cell junction is the adherens junction. This type of junction usually forms a subapical, beltlike structure that mechanically links the lateral surfaces of adjacent epithelial cells. Adherens junctions contain transmembrane proteins of the cadherin family that mediate homotypic adhesion. Cadherins are thought to connect to the actin cytoskeleton indirectly through the proteins α-catenin and β-catenin. Catenin–cadherin complexes also are associated with sites of contact between blastomeres in vertebrate and invertebrate embryos. In Drosophila, mice, and Xenopus, gene inactivation of catenins or cadherins disrupts general cell adhesion and apicobasal polarity of blastomeres and epithelial cells (Heasman et al., 1994; Larue et al., 1994; Haegel et al., 1995; Cox et al., 1996; Müller and Wieschaus, 1996; Kafron et al., 1997; Torres et al., 1997). Thus, it has been difficult to define direct requirements for these proteins in cytoskeletal organization and morphogenesis, although there is evidence for specific roles in tracheal cell migration (Tanaka-Matakatsu et al., 1996) and axon outgrowth (Iwai et al., 1997) in Drosophila.The Caenorhabditis elegans embryo provides a model system for studying how cells move and change shape to generate body and tissue morphologies. At hatching, the outermost cellular layer of the body consists of a monolayer of 85 epithelial cells called hypodermal cells that are linked together by adherens junctions (White, 1988). During embryogenesis, hypodermal cells are involved in two distinct processes that transform the initially ellipsoidal mass of embryonic cells into a long, thin worm; these processes are called body enclosure and body elongation (Sulston et al., 1983; Priess and Hirsh, 1986; Williams-Masson et al., 1997). The hypodermal cells are born on the dorsal surface of the embryo. As the hypodermal cells develop adherens junction connections, they begin to spread as a sheet across the embryo until the contralateral edges of the sheet meet at the ventral midline. In the anterior of the embryo, ventral hypodermal cells on the periphery of the spreading sheet develop filopodial extensions that may function to draw the contralateral edges of the sheet together (Williams-Masson et al., 1997). In the posterior of the embryo, the contralateral edges appear to be drawn together by a purse-string–like contraction that completes the enclosure process (Williams-Masson et al., 1997). In several respects, these processes are similar to epithelial cell movements described in a variety of systems, such as wound healing in vertebrates (Martin and Lewis, 1992) and dorsal closure in Drosophila (Young et al., 1993). At the completion of body enclosure in C. elegans, the apical surfaces of the hypodermal cells resemble rectangles that are elongated along the circumferential contour of the embryo''s body. These apical surfaces begin to change shape, constricting along the circumferential contour of the body and elongating along the anterior–posterior (longitudinal) axis. The coordinate changes in the shapes of the hypodermal cells appear to cause the body to decrease in circumference and to elongate about fourfold along its longitudinal axis (Sulston et al., 1983; Priess and Hirsh, 1986). Before body elongation, the apical cytoskeleton of each hypodermal cell reorganizes to form an array of parallel actin filament bundles oriented along the circumferential contour of the body (Priess and Hirsh, 1986; Costa et al., 1997). The parallel filament bundles bridge two opposing sides of each hypodermal cell, apparently connecting to the subapical adherens junction. Contraction of the filament bundles has been proposed as the force that elongates the embryo; the bundles become shorter and thicker during elongation, and drugs that disrupt actin filament organization prevent elongation. Apical constriction of cells has been shown in other systems to drive the invagination of epithelial sheets; because of the closed, cylindrical geometry of the hypodermal sheet in C. elegans, an analogous apical constriction might instead drive body elongation (Priess and Hirsh, 1986). Although the morphology and properties of the hypodermal cells strongly suggest that they mediate body elongation, almost all of the elongation-defective mutants described thus far have mutations in genes encoding muscle or basement membrane components. Body-wall muscles underlie the hypodermis, separated by a basement membrane (Hresko et al., 1994; diagram in Fig. ​Fig.88 a). Mutations in any of several genes that eliminate embryonic muscle contraction prevent elongation beyond a twofold increase in body length; this phenotype is called Pat¹ (paralyzed, arrested elongation at twofold; Williams and Waterston, 1994). Some of the genes of the Pat class have been shown to encode muscle-specific proteins. Because the muscles and myofilaments are oriented longitudinally, muscle contraction would be expected to oppose body elongation; thus, it is not yet understood why muscle function is required for complete elongation. The genes let-2 and emb-9 encode basement membrane collagens, and mutations in these genes produce elongation defects similar to those of Pat mutants (Guo et al., 1991; Sibley et al., 1993; Williams and Waterston, 1994). The only gene identified that is both required for proper body elongation and apparently expressed in hypodermal cells is let-502 (Wissmann et al., 1997). The predicted LET-502 protein is related to Rho-binding kinases, which can activate myosin light chain kinase, suggesting that LET-502 could have a role in hypodermal cells for the contraction of the array of actin filament bundles. Open in a separate windowFigure 8Models of morphogenetic forces and molecular organization at hypodermal cell junctions. (A) Oblique view of a schematic cross-section of an embryo after fusion of the dorsal hypodermal cells. CFBs are shown as thin lines, and adherens junctions are shown as thick lines. Bands of longitudinally oriented body wall muscles (m) are shown underlying the hypodermal cells. (B) Mechanical model of forces between the dorsal hypodermis and a lateral hypodermal cell. The connection between each CFB and the adherens junction (AJ) is represented as an open circle. In the lateral hypodermal cell, the connections are pulled downward by contraction of the CFBs within the lateral cell itself and pulled upward as CFBs in the dorsal cell contract. Note that the adherens junction at the two ends of the lateral cell (shown as two springs) are oriented such that they could dissipate some of the force exerted by contractions in the dorsal cell. (C) Two molecular models for the linkage between a filament (CF) in a CFB to a filament (AJF) in the adherens junction. HMR-1 is shown at the membrane contacts between two cells and associated with HMP-2. In the top cell, HMP-1 links a CF and an AJF directly; in the bottom cell, HMP-1 links different AJFs together while another factor (X) provides the link between the AJFs and CFs. To expand our understanding of the molecular basis for morphogenesis, we have isolated and characterized a group of mutants that display similar defects in embryo morphogenesis. In this paper, we present evidence that a C. elegans catenin–cadherin system mediates morphogenetic cell shape changes and specific aspects of cytoskeletal organization. We show that the genes hmp-1, hmp-2, and hmr-1 are required for the proper migration of hypodermal cells during body enclosure and for body elongation. We demonstrate that hmp-1, hmp-2, and hmr-1 can encode proteins related to α-catenin, β-catenin, and cadherin, respectively. We show that the protein products of these genes are localized to adherens junctions in the hypodermis. Our results indicate that these proteins anchor the parallel actin filament bundles to the adherens junctions in hypodermal cells and that this coupling translates the force of bundle contraction into cell shape change.     

Aio-Casilio: a robust CRISPR–Cas9–Pumilio system for chromosome labeling

The biological function of chromatin bases on its spatial organization and dynamic activities in different situations. Labeling and tracing of genomic sequences has been a huge challenge in studying the spatial dynamics of chromatin. We reported the development of ‘all-in-one Casilio system (Aio-Casilio)’, a new system that enables the labeling of endogenous genomic loci. The Aio-Casilio system consists of the dCas9 protein, mClover fused with the Pumilio and FBF proteins RNA-binding domain (PUF domain) and an U6-sgRNA appended with multiple PUF-binding site(s). Here we showed that the Aio-Casilio system is robust tool for imaging of repetitive elements in telomeres and major satellite in N2A cells. Furthermore, we developed a PBTon–Aio-Casilio System, which enables the visualization of repetitive sequences in mES cells. However, this system failed to establish a labeled ES cell line when we attempted to establish a stable labeling cell line for living cell image. This Aio-Casilio imaging tool has potential to significantly improve the capacity to study the conformation of chromosomes in living cells.     

CRISPR/Cas9: a promising way to exploit genetic variation in plants

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.     

CRISPR–Cas system: a powerful tool for genome engineering

Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Recent advances with the RNA-mediated Cas9 endonuclease derived from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems have dramatically transformed our ability to specifically modify intact genomes of diverse cells and organisms. The CRISPR–Cas system has been adapted as an efficient, facile, and robust gene-targeting technology with the potential for high-throughput and multiplexed genome engineering. Exciting breakthroughs in understanding the mechanisms of the CRISPR–Cas system and its enormous potential for applications across basic science, agricultural and biotechnology.     

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR–Cas9 cassette or a transient CRISPR–Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.     

CRISPR–Cas9D10A nickase-assisted base editing in the solvent producer Clostridium beijerinckii

Clostridium beijerinckii is a potentially important industrial microorganism as it can synthesize valuable chemicals and fuels from various carbon sources. The establishment of convenient to use, effective gene tools with which the organism can be rapidly modified is essential if its full potential is to be realized. Here, we developed a genomic editing tool (pCBEclos) for use in C. beijerinckii based on the fusion of cytidine deaminase (Apobec1), Cas9 ^D10A nickase and uracil DNA glycosylase inhibitor (UGI). Apobec1 and UGI are guided to the target site where they introduce specific base-pair substitutions through the conversion of C·G to T·A. By appropriate choice of target sequence, these nucleotide changes are capable of creating missense mutation or null mutations in a gene. Through optimization of pCBEclos, the system derived, pCBEclos-opt, has been used to rapidly generate four different mutants in C. beijerinckii, in pyrE, xylR, spo0A, and araR. The efficiency of the system was such that they could sometimes be directly obtained following transformation, otherwise only requiring one single restreaking step. Whilst CRISPR–Cas9 nickase systems, such as pNICKclos2.0, have previously been reported in C. beijerinckii, pCBEclos-opt does not rely on homologous recombination, a process that is intrinsically inefficient in clostridia such as C. beijerinckii. As a consequence, bulky editing templates do not need to be included in the knockout plasmids. This both reduces plasmid size and makes their construction simpler, for example, whereas the assembly of pNICKclos2.0 requires six primers for the assembly of a typical knockout plasmid, pCBEclos-opt requires just two primers. The pCBEclos-opt plasmid established here represents a powerful new tool for genome editing in C. beijerinckii, which should be readily applicable to other clostridial species.     

Exploiting CRISPR–Cas immune systems for genome editing in bacteria

1. Download : Download high-res image (131KB)
2. Download : Download full-size image