Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex

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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.     

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F₁. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species.     

Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system

The mammalian zygote-mediated CRISPR/Cas system can efficiently generate targeted genome-modified animals. However, this system is limited by the risk of off-target mutations. Here we show that offset-nicking by Cas9 nickase and paired gRNAs allows us to generate region deleted mice and targeted knock-in mice without off-target mutations.     

A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval–pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.