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Genome editing efficiency of four Drosophila suzukii endogenous U6 promoters.
The invasive spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) has caused serious economic losses to the fruit industry. The conventional control methods have many limitations and genetic engineering technologies such as CRISPR/Cas9-mediated gene drive are promising approaches. In the CRISPR/Cas9 system, the transcriptional regulatory elements play an important role in the activities of gRNA. Thus, in order to improve the genome editing efficiency of the CRISPR/Cas9 system in D. suzukii, we cloned and tested four endogenous U6 promoters to drive mutagenesis of the white gene. Our results showed that all the four promoters could be used with variable efficiency. The promoter DsU6-3 had the highest genome editing efficiency among the four DsU6 promoters. Compared with the DsU6-3 promoter, the DmU6:3 promoter showed lower efficiency to drive mutagenesis in D. suzukii. These findings expand the range of promoters available to express gRNAs in D. suzukii, facilitating the basic and applied research on this important pest.
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Isogenic human SNCA gene dosage induced pluripotent stem cells to model Parkinson's disease.
Alpha-synuclein overexpression and aggregation are critical factors in the pathogenesis of Parkinson's disease (PD). Clinical cases with alpha-synuclein (SNCA) multiplications or deletions indicate that gene expression levels are essential for neurodegeneration and neurodevelopment. Here, we developed an isogenic SNCA gene dosage model using CRISPR/Cas9 gene editing to introduce frameshift mutations into exon 2 of the SNCA coding region in human induced pluripotent stem cells (iPSCs) from a patient with an SNCA triplication. We derived and characterized clones with different frameshift mutations. This isogenic SNCA gene dosage panel will address the physiological and detrimental effects of varying alpha-synuclein expression levels.
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In Vitro Correction of Point Mutations in the DYSF Gene Using Prime Editing.
Dysferlinopathy is caused by over 500 mutations in the gene encoding dysferlin, including close to 300 point mutations. One option to cure the disease is to use a gene therapy to correct these mutations at the root. Prime editing is a technique which can replace the mutated nucleotide with the wild-type nucleotide. In this article, prime editing is used to correct several point mutations in the DYSF gene responsible for dysferlinopathy. In vitro editing of HEK293T cells reaches up to 31%. Notably, editing was more efficient in myoblasts than in patient-derived fibroblasts. The prime editing technique was also used to create a new myoblast clone containing a patient mutation from a healthy myoblast cell line.
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Targeted genome regulation via synthetic programmable transcriptional regulators.
Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications.
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Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.
In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.
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Hypersymbiotics™: An artistic reflection on the ethical and environmental implications of microbiome research and new technologies.
This essay describes my ongoing series "Hypersymbiotics™," which began in 2012 and explores the potential ways in which our microbiome, genetics, epigenetics and even our environment could potentially be enhanced to turn us into human 'super-organisms.' The series includes performances and installations involving BioArt, as well as photographic documentation of ephemeral artworks and takes the form of a vehicle for public discussion about new healthcare technologies. The essay discusses artworks made using synthetic biology techniques including CRISPR genetic modification in bacteria and yeasts, and gene editing in plants, as well as using artificial intelligence and stem cell research. It critiques the role of the media and advertising in the promotion of complex new biomedical technologies. The "Hypersymbiotics™" series is deeply concerned with promoting public understanding of the ethical implications of new scientific developments and enabling reflection and debate. At its core the artwork is about knowledge, power, and control and where that resides.
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Off-target genome editing: A new discipline of gene science and a new class of medicine.
With an increasing growth of genome editing, off-target effects such as non-specific genetic modifications resulting from the designed process of genome editing become a new discipline of gene science and new class medicine. The degree of short-term and long-term side effects and toxicity or dynamics of the primary and secondary off-target genome editing varies with the application of different methodologies of gene editing and measuring, readouts of genetic modifications, or comparison reference. Measurements of dynamic off-target effects caused directly or indirectly by genome editing are critical in clinical application of gene editing. The quality of genome editing methods is one of the decisive factors in the occurrence of off-target effects. Mechanisms by which off-target effects of genome editing occurs are more complex and comprehensive than we expected. The heterogeneity of off-target effects of gene-edited cells at single-cell levels should be defined during the development and formation of cell clusters. In addition to off-target effects on gene-edited cells per se, alterations of gene sequence, structure, dimension, and function of related regulators caused by off-target effects may also influence intercellular communications and interactions between gene-edited cells, between gene-edited cells and non-edited cells, or between non-edited cells. Thus, controlling, measuring, defining, categorizing, and predicting off-target genome editing need to be standardized and prioritized before clinical application of gene editing.
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Advances in CRISPR Technologies for Microbial Strain Engineering.
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Embryo editing takes another step to clinic.
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Viral Vectors, Engineered Cells and the CRISPR Revolution.
Over the past few decades the ability to edit human cells has revolutionized modern biology and medicine. With advances in genome editing methodologies, gene delivery and cell-based therapeutics targeted at treatment of genetic disease have become a reality that will become more and more essential in clinical practice. Modifying specific mutations in eukaryotic cells using CRISPR-Cas systems derived from prokaryotic immune systems has allowed for precision in correcting various disease mutations. Furthermore, delivery of genetic payloads by employing viral tropism has become a crucial and effective mechanism for delivering genes and gene editing systems into cells. Lastly, cells modified ex vivo have tremendous potential and have shown effective in studying and treating a myriad of diseases. This chapter seeks to highlight and review important progress in the realm of the editing of human cells using CRISPR-Cas systems, the use of viruses as vectors for gene therapy, and the application of engineered cells to study and treat disease.
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