CRISPR/Cas9‐mediated gene knockout in the ascidian Ciona intestinalis

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.     

CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass

Genetic engineering in livestock has been greatly enhanced through the use of artificial programmed nucleases such as the recently emerged clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system. We recently reported our successful application of the CRISPR/Cas9 system to engineer the goat genome through micro‐injection of Cas9 mRNA and sgRNAs targeting MSTN and FGF5 in goat embryos. The phenotypes induced by edited loss‐of‐function mutations of MSTN remain to be evaluated extensively. We demonstrate the utility of this approach by disrupting MSTN, resulting in enhanced body weight and larger muscle fiber size in Cas9‐mediated gene‐modified goats. The effects of genome modifications were further characterized by H&E staining, quantitative PCR, Western blotting and immunofluorescence staining. Morphological and genetic analyses indicated the occurrence of phenotypic and genotypic modifications. We further provide sufficient evidence, including breeding data, to demonstrate the transmission of the knockout alleles through the germline. By phenotypic and genotypic characterization, we demonstrated the merit of using the CRISPR/Cas9 approach for establishing genetically modified livestock with an enhanced production trait.     

CRISPR/Cas9‐induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock‐in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near‐maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.     

CRISPR/Cas9‐mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni

The Queensland fruit fly, Bactrocera tryoni (Froggatt), is a polyphagous horticultural pest in Australia that is capable of causing significant damage to more than 100 different host fruits and vegetables. Chemical applications and ecological control strategies, such as the sterile insect technique (SIT), are commonly used to suppress established populations and eradicate invasive outbreaks following migration. The recently published B. tryoni draft genome provides new opportunities to identify candidate genes for targeted genome modification in order to generate advanced genetic strains for management using sterile insect strategies. Here, we demonstrate CRISPR/Cas‐mediated mutagenesis in B. tryoni through generating a series of frame‐shift mutations in the ATP‐dependent binding cassette transporter, white, causing a classic white‐eye phenotype. This work establishes methods for CRISPR/Cas genome editing in tephritids and demonstrates its potential for developing genetic sexing strains which could be used for SIT‐based pest control.     

Expanding the scope of CRISPR/Cas9‐mediated genome editing in plants using an xCas9 and Cas9‐NG hybrid

The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome‐editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9‐NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9‐NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG‐Cas9) based on mutations from both xCas9 and Cas9‐NG, and found that XNG‐Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG‐Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9‐NG) active in plant.     

CRISPR/Cas9‐mediated genome modification in the mollusc,Crepidula fornicata

The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata, and have successfully created transgenic embryos expressing mCherry fused to endogenous β‐catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β‐catenin‐specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock‐in strategy permits in vivo observations of β‐catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9‐mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. genesis 53:237–244, 2015. © 2014 Wiley Periodicals, Inc.     

Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843. © 2013 Wiley Periodicals, Inc.     

Engineering of CRISPR/Cas9‐mediated potyvirus resistance in transgene‐free Arabidopsis plants

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence‐specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field‐grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene‐free T₂ generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T₃ lines revealed no differences from wild‐type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.     

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.     

Fast and cloning‐free CRISPR/Cas9‐mediated genomic editing in mammalian cells

CHoP‐In (CRISPR/Cas9‐mediated Homology‐independent PCR‐product integration) is a fast, non‐homologous end‐joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co‐transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon‐homologous end joining. The approach is versatile, allowing N‐terminal, C‐terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands‐on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.     

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.     

Design of a bacterial speck resistant tomato by CRISPR/Cas9‐mediated editing of SlJAZ2

Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade‐off is a major challenge for obtaining broad‐spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co‐receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by PtoDC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co‐receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C‐terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to PtoDC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade‐off in a crop, by spatially uncoupling the SA‐JA hormonal antagonism at the stomata, entry gates of specific microbes such as PtoDC3000. Moreover, our results also constitute a novel CRISPR/Cas‐based strategy for crop protection that could be readily implemented in the field.     

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.     

Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in Solanum lycopersicum

In recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.