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[A perspective on the application of CRISPR/CAS9 genome editing system to study of molecular-genetic basis of mental disorders].
Schizophrenia, depression, bipolar disorder and autism spectrum disorders are common mental disorders that are among the leading causes of disability worldwide. The major complication to effective therapies for mental disorders is the poor understanding of their pathogenic mechanisms. Currently, an increasing number of research groups are focusing on uncovering the molecular mechanisms of mental disorders and developing novel therapies using the CRISPR/Cas9 (Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) - CRISPR-associated system 9 (Cas9)) system to determine the molecular mechanisms of developing mental disorders and novel therapy. The CRISPR/Cas9 system is the most promising among genome editing tools. Numerous advantages of the CRISPR/Cas9 system and its successful application in some studies provide wide opportunities for genome therapy and regeneration medicine. In this review we shortly describe structure and function of the CRISPR/Cas9 system and its application to study the molecular-genetic basis of mental disorders in human.
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Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System.
The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min-1 and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.
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The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.
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Current progress of genome editing in livestock.
Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.
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Emergent CRISPR-Cas-based technologies for engineering non-model bacteria.
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) technologies brought a transformative change in the way bacterial genomes are edited, and a plethora of studies contributed to developing multiple tools based on these approaches. Prokaryotic biotechnology benefited from the implementation of such genome engineering strategies, with an increasing number of non-model bacterial species becoming genetically tractable. In this review, we summarize the recent trends in engineering non-model microbes using CRISPR-Cas technologies, discussing their potential in supporting cell factory design towards biotechnological applications. These efforts include, among other examples, genome modifications as well as tunable transcriptional regulation (both positive and negative). Moreover, we examine how CRISPR-Cas toolkits for engineering non-model organisms enabled the exploitation of emergent biotechnological processes (e.g. native and synthetic assimilation of one-carbon substrates). Finally, we discuss our slant on the future of bacterial genome engineering for domesticating non-model organisms in light of the most recent advances in the ever-expanding CRISPR-Cas field.
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Gene Therapy for Inherited Retinal Degeneration.
Inherited retinal degeneration (IRD), a group of rare retinal diseases that primarily lead to the progressive loss of retinal photoreceptor cells, can be inherited in all modes of inheritance: autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), and mitochondrial. Based on the pattern of inheritance of the dystrophy, retinal gene therapy has 2 main strategies. AR, XL, and AD IRDs with haploinsufficiency can be treated by inserting a functional copy of the gene using either viral or nonviral vectors (gene augmentation). Different types of viral vectors and nonviral vectors are used to transfer plasmid DNA both in vitro and in vivo. AD IRDs with gain-of-function mutations or dominant-negative mutations can be treated by disrupting the mutant allele with (and occasionally without) gene augmentation. This review article aims to provide an overview of ocular gene therapy for treating IRDs using gene augmentation with viral or nonviral vectors or gene disruption through different gene-editing tools, especially with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system.
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Cas9-based genome editing in Drosophila.
Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.
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Immune responses to CRISPR-Cas protein.
The clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) protein technologies have evolved as promising, cost-effective, and efficient methods for editing genomes. Editing genomes with high specificity and precision is a daunting task, where errors can lead to undesirable outcomes. Many elegant studies have successfully shown that the CRISPR-Cas9 system can modify, disrupt, and add new DNA sequences directly into the genomes of the cells or animals being studied. As such, the CRISPR-Cas9 technology holds immense potential for biomedical research as well as agricultural and therapeutic applications, further emphasized by its unprecedented movement into the clinical setting. Throughout every stage of life, missense mutations can lead to highly unfavorable outcomes, syndromes, and diseases. Many of these mutations are transferred directly through the fertilization process and, thereby, acquired at birth and propagated to the next generation. As such, it has been of great interest to develop techniques to repair these mutations using genetic manipulation, prior to or following birth. CRISPR-Cas9 has many advantages in this regard over numerous other existing technologies. Regardless, editing bases within a genome can be associated with numerous challenges that were previously unrecognized and lead to unforeseen consequences. While the CRISPR-Cas9 method is perfectly suitable for editing cells outside the body with limited risk to the normal functioning of the cell, recent publications have illustrated a number of challenging conditions resulting from its use. One of them is directed to the host immune response toward CRISPR-Cas9. With this in mind, this review will discuss recent observations on the host immune response to CRISPR-Cas9 and the associated challenges that arise as a result.
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Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos.
The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.
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CRISPR-based genome editing in primary human pancreatic islet cells.
Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.
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