Genome Editing Techniques Research Articles

Genomics and genome editing techniques of crickets, an emerging model insect for biology and food science.

Most tools available for manipulating gene function in insects have been developed for holometabolous species. In contrast, functional genetics tools for the Hemimetabola are highly underdeveloped. This is a barrier both to understanding ancestral insect biology, and to optimizing contemporary study and manipulation of particular large hemimetabolous orders of crucial economic and agricultural importance like the Orthoptera. For orthopteran insects, including crickets, the rapid spread of next-generation sequencing technology has made transcriptome data available for a wide variety of species over the past decade. Furthermore, whole genome sequences of orthopteran insects with relatively large genome sizes are now available. With these new genome assemblies and the development of genome editing technologies such as the CRISPR-Cas9 system, it has become possible to create gene knock-out and knock-in strains in orthopteran insects. As a result, orthopteran species should become increasingly feasible for laboratory study not only in research fields that have traditionally used insects, but also in agricultural fields that use them as food and feed. In this review, we summarize these recent advances and their relevance to such applications.

Flexible auditory training, psychophysics, and enrichment of common marmosets with an automated, touchscreen-based system

Devising new and more efficient protocols to analyze the phenotypes of non-human primates, as well as their complex nervous systems, is rapidly becoming of paramount importance. This is because with genome-editing techniques, recently adopted to non-human primates, new animal models for fundamental and translational research have been established. One aspect in particular, namely cognitive hearing, has been difficult to assess compared to visual cognition. To address this, we devised autonomous, standardized, and unsupervised training and testing of auditory capabilities of common marmosets with a cage-based standalone, wireless system. All marmosets tested voluntarily operated the device on a daily basis and went from naïve to experienced at their own pace and with ease. Through a series of experiments, here we show, that animals autonomously learn to associate sounds with images; to flexibly discriminate sounds, and to detect sounds of varying loudness. The developed platform and training principles combine in-cage training of common marmosets for cognitive and psychoacoustic assessment with an enriched environment that does not rely on dietary restriction or social separation, in compliance with the 3Rs principle.

Implementing CRISPR-Cas9 Yeast Practicals into Biology Curricula.

CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast, Saccharomyces cerevisiae. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.

A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants.

BackgroundThe genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.ResultsHere, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.ConclusionsOur results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants.

Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200–250 nucleotides long sequence to either 5′ or 3′ ends of guide RNA did not significantly affect Cas9 cleavage activity.

EP090: Precision medicine modelling for undiagnosed and rare disease

Despite advances in exome, genome, and RNA sequencing, up to 60-70% of individuals with suspected genetic disorders remain undiagnosed likely because their pathogenic variants have not yet been discovered. Alternatively, some individuals may remain undiagnosed because the clinical significance of identified variants is unclear. The Undiagnosed Diseases Network (UDN) Model Organism Screening Center (MOSC) has successfully utilized various genome editing techniques in the fly, worm and zebrafish to model human variants and provide data supporting the interpretation of variants of uncertain significance.

English

The efficient plantlets regeneration of peanut is the most important and a primary step to successfully transform gene and apply recently developed genome editing techniques for crop yield improvement. The purpose of this research is to develop protocol for peanut cultivars applying different concentration of hormones for selected peanut cultivars and develop plantlets regeneration protocol. There is no previously reported protocol for the Chinese peanut cultivar N3 and Yu-hua-14. We optimized shoot and root regeneration protocol for two peanut cultivars. Both cultivars showed positive response for the cytokinin plant growth hormone 6-benzylaminopurine (BAP) and thidiazuron (TDZ). The highest shooting rate (97%) was found in a medium supplemented with 4 mg/L BAP and (94.33%) for 1 mg/L TDZ. Hence, more shoot initiation was observed at higher concentration of BAP as compared to TDZ. However, the maximum root regeneration (81%) was found on medium containing 0.3 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D) and the highest rooting rate (96.33%) was found in a medium containing 1 mg/L α-naphthalene acetic acid (NAA), indicating lower concentration of NAA induce more rooting rate than 2,4-D treatment. In this study, cotyledonary node was used, and this method was found to be efficient and rapid for in vitro peanut regeneration. © 2022 Friends Science Publishers

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genome-editing toolkit to enhance salt stress tolerance in rice and wheat.

The rice and wheat agricultural system is the primary source of food for billions across the world. However, the productivity and long-term sustainability of rice and wheat are threatened by a large number of abiotic stresses, especially salinity stress. Salinity has a significant impact on plant development and productivity and is one of the leading causes of crop yield losses in agricultural soils worldwide. Over the last few decades, several attempts have been undertaken to enhance salinity stress tolerance, most of which have relied on traditional or molecular breeding approaches. These approaches have so far been insufficient in addressing the issues of abiotic stress. However, due to the availability of genome sequences for cereal crops like rice and wheat and the development of genome editing techniques like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9), it is now possible to "edit" genes and influence key traits. Here, we review the application of the CRISPR/Cas9 system in both rice (Oryza sativa L.) and wheat (Triticum aestivum L.) to develop salinity tolerant cultivars. The CRISPR/Cas genome editing toolkit holds great promise of producing cereal crops tolerant to salt stress to increase agriculture resilience with a strong impact on the environment and public health.

Mutation-specific reporter for optimization and enrichment of prime editing

Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

A link between DNA double-strand breaks and regulation of global translation.

In response to double-strand breaks (DSBs) in the DNA, cells undergo transcriptional, translational and post-translational reprogramming to tackle the damage. In this study by Riepe etal., the authors have shown that the global translation inhibition of proteins is concomitant to DNA damage response. Treatment with various DSB-generating agents can cause a major downregulation in the translation of cellular proteins except for the ISR (integrated stress response) proteins. Authors report a specific and significant reduction in the level of a core ribosomal RPS27A protein coupled to kinetics of DSB induction and repair. The study proposes that molecular alterations generated as a by-product of DNA damage may inadvertently impact phenotypic responses of the cells and a cautious approach must be followed when utilizing DSB-based genome editing techniques. Comment on: https://doi.org/10.1111/febs.16321.

PYL1- and PYL8-like ABA Receptors of Nicotiana benthamiana Play a Key Role in ABA Response in Seed and Vegetative Tissue.

To face the challenges of climate change and sustainable food production, it is essential to develop crop genome editing techniques to pinpoint key genes involved in abiotic stress signaling. The identification of those prevailing abscisic acid (ABA) receptors that mediate plant-environment interactions is quite challenging in polyploid plants because of the high number of genes in the PYR/PYL/RCAR ABA receptor family. Nicotiana benthamiana is a biotechnological crop amenable to genome editing, and given the importance of ABA signaling in coping with drought stress, we initiated the analysis of its 23-member family of ABA receptors through multiplex CRISPR/Cas9-mediated editing. We generated several high-order mutants impaired in NbPYL1-like and NbPYL8-like receptors, which showed certain insensitivity to ABA for inhibition of seedling establishment, growth, and development of shoot and lateral roots as well as reduced sensitivity to the PYL1-agonist cyanabactin (CB). However, in these high-order mutants, regulation of transpiration was not affected and was responsive to ABA treatment. This reveals a robust and redundant control of transpiration in this allotetraploid plant that probably reflects its origin from the extreme habitat of central Australia.

Chromosomal Engineering through CRISPR– Cas Technology: A Way Forward

The breeding of crops is dependent on the potential to interrupt or maintain genetic links between characteristics, and the availability of genetic variability. CRISPR-Cas is a new genome-editing technique that has made it possible for breeders to introduce regulated and site-specific genetic diversity while simultaneously improving qualities with high efficacy. The existence of genomic linkage is a barrier in transferring desirable features among domesticated species from their wild counterparts. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Consequently, attempts have been made to induce site-specific DSBs in both somatic and meiotic plant cells by utilizing CRISPR–Cas techniques to achieve preset crossovers among homologs. Nonetheless, this method has not yielded significant heritable homologous crossings which were recombination-based. Lately, CRISPR–Cas has been used to achieve hereditary chromosomal rearrangements (CRs), including translocations and inversions, in plants. This method allows for the development of megabase CRs by DSB repair through non-homologous end-joining after insertion of DSBs in somatic plant cells. This technique may potentially make it possible to restructure genomes on a more global scale, culminating in the creation not just of synthetic plant chromosomes, but also that of new plant species.

Gene editing and its applications in biomedicine.

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.

Current Status of CRISPR/Cas9 Application in Clinical Cancer Research: Opportunities and Challenges.

Simple SummaryIt is widely believed that cancer is developed due to changes in the genetic codes of our DNA, leading to abnormal growth of cells. In the past few years scientists have discovered a system which is used as an immune mechanism by bacteria in order to cleave the invading viruses, called CRISPR/Cas9. Exploiting this system in humans will allow scientists to attempt to edit genetic errors that lead to cancer. This scientific breakthrough has a lot of potential for treating a variety of diseases, including cancer. It has already been successfully used in treatment of some types of blood cancer. In this article, we discuss the opportunities and limitations of CRISPR/Cas9 in treatment of solid cancer.Cancer is considered by not only multiple genetic but also epigenetic amendments that drive malignant cell propagation and consult chemo-resistance. The ability to correct or ablate such mutations holds enormous promise for battling cancer. Recently, because of its great efficiency and feasibility, the CRISPR-Cas9 advanced genome editing technique has been extensively considered for therapeutic investigations of cancers. Several studies have used the CRISPR-Cas9 technique for editing cancer cell genomic DNA in cells and animal cancer models and have shown therapeutic potential in intensifying anti-cancer protocols. Moreover, CRISPR-Cas9 may be used to correct oncogenic mutations, discover anticancer drugs, and engineer immune cells and oncolytic viruses for immunotherapeutic treatment of cancer. We herein discuss the challenges and opportunities for translating therapeutic methods with CRISPR-Cas9 for clinical use and suggest potential directions of the CRISPR-Cas9 system for future cancer therapy.

Drought tolerance improvement in Solanum lycopersicum: an insight into "OMICS" approaches and genome editing.

Solanum lycopersicum (tomato) is an internationally acclaimed vegetable crop that is grown worldwide. However, drought stress is one of the most critical challenges for tomato production, and it is a crucial task for agricultural biotechnology to produce drought-resistant cultivars. Although breeders have done a lot of work on the tomato to boost quality and quantity of production and enhance resistance to biotic and abiotic stresses, conventional tomato breeding approaches have been limited to improving drought tolerance because of the intricacy of drought traits. Many efforts have been made to better understand the mechanisms involved in adaptation and tolerance to drought stress in tomatoes throughout the years. "Omics" techniques, such as genomics, transcriptomics, proteomics, and metabolomics in combination with modern sequencing technologies, have tremendously aided the discovery of drought-responsive genes. In addition, the availability of biotechnological tools, such as plant transformation and the recently developed genome editing system for tomatoes, has opened up wider opportunities for validating the function of drought-responsive genes and the generation of drought-tolerant varieties. This review highlighted the recent progresses for tomatoes improvement against drought stress through "omics" and "multi-omics" technologies including genetic engineering. We have also discussed the roles of non-coding RNAs and genome editing techniques for drought stress tolerance improvement in tomatoes.

Prevention of Enzymatic Browning by Natural Extracts and Genome-Editing: A Review on Recent Progress.

Fresh fruits and vegetable products are easily perishable during postharvest handling due to enzymatic browning reactions. This phenomenon has contributed to a significant loss of food quality and appearance. Thus, a safe and effective alternative method from natural sources is needed to tackle enzymatic browning prevention. The capabilities of natural anti-browning agents derived from plant- and animal-based resources in inhibiting enzymatic activity have been demonstrated in the literature. Some also possess strong antioxidants properties. This review aims to summarize a recent investigation regarding the use of natural anti-browning extracts from different sources for controlling the browning. The potential applications of genome-editing in preventing browning activity and improving postharvest quality is also discussed. Moreover, the patents on the anti-browning extract from natural sources is also presented in this review. The information reviewed here could provide new insights, contributing to the development of natural anti-browning extracts and genome-editing techniques for the prevention of food browning.

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

SummaryThe pantry moth Plodia interpunctella is a worldwide pest of stored food products and a promising laboratory model system for lepidopteran functional genomics. Here we describe efficient methods for precise genome editing in this insect. A spontaneous recessive white-eyed phenotype maps to a frameshift deletion (c.737delC) in the white gene. CRISPR NHEJ mutagenesis of white replicates this phenotype with high rates of somatic biallelic knockout. G0 individuals with mutant clones on both eyes produced 100% mutant progeny, making white an ideal marker for co-conversion when targeting other genes. CRISPR HDR experiments corrected c.737delC and reverted white eyes to a pigmented state in 37% of G0 mosaic adults. These repaired alleles showed practical rates of germline transmission in backcrosses, demonstrating the potential of the technique for precise genome editing. Plodia offers a promising avenue for research in this taxon because of its lab-ready features, egg injectability, and editability.

Ketamine's fast-acting antidepressant mechanisms: state of the art and new research perspectives

About a third of patients with major depressive disorder (MDD) do not have an adequate response to first-line antidepressant treatment, i.e., develop a treatment-resistant depression (TRD). The partial understanding of MDD pathophysiology currently constitutes the major barrier to clinical and research progress on this topic. However, recent advances in genome editing techniques as well as in induced pluripotent stem cells (iPSC) technology are offering unprecedented opportunities in both human disease modelling and drug discovery. These technology progresses have been enabling to set up disease-relevant patient-specific in vitro disease modeling for various mental disorders. The resulting models have the potential to significantly improve pathophysiologic understanding of MDD and then overcome some limitations inherent to animal and post-mortem models. More recently, psychiatry started to deal with the fast acting antidepressant ketamine and its derivates. Although ketamine appears to have the potential to transform the treatment of depression, its specific mechanisms of action are only partially known. Such knowledge is necessary to develop a model to understand the mechanisms behind fast-acting antidepressants, which may enable the discovery of novel glutamatergic compounds for the treatment of MDD. After discussing both the current understanding of ketamine's mechanisms of action, and the state of the art of human iPSC technology, the authors will introduce the implementation of a TRD model based on iPSC human technology and aimed at studying the ketamine's fast acting antidepressant mechanisms of action.

Deciphering the Fragmentation of the Human Genome Editing Regulatory Landscape

Genome editing techniques have generated a growing interest following the discovery of the so-called CRISPR-Cas technique. It has raised a global uproar as regards its use in humans, especially after the 2018 announcement of a Chinese scientist who had used CRISPR to edit the genes of twin embryos. Indeed, one of the greatest concerns, although not the only one, has been the use of genome editing technologies to modify the human germline. In such scientific and technological context, the law plays a key role in framing what should be allowed or prohibited, and under which conditions, to find a balance between safe and accessible innovative treatments and respect of fundamental rights in accordance with the societal values and choices. Within the European Union, several institutions have considered the issues raised by human genome editing, and several legal texts participate in the establishment of the European regulatory framework applicable to human genome editing. Yet we argue in this article that the established regulatory landscape is fragmented in the sense of being divided, split, or segmented. Such fragmentation, which may have been inevitable for historical and technicolegal reasons, produces effects regarding the role of the current regulatory frameworks applicable to human genome editing. Focusing on the European Union and on the French levels of governance, we discuss how such fragmentation takes place through the identification of determinants of the human genome editing fragmented regulatory landscape. We argue that it should be seen as a process providing more contingent responses to human genome editing reflecting changing political and legal contexts.