Development Of Genome Editing Techniques Research Articles

Harnessing CRISPR-Cas for oomycete genome editing.

Oomycetes are a group of microorganisms that include pathogens responsible for devastating diseases in plants and animals worldwide. Despite their importance, the development of genome editing techniques for oomycetes has progressed more slowly than for model microorganisms. Here, we review recent breakthroughs in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technologies that are expanding the genome editing toolbox for oomycetes - from the original Cas9 study to Cas12a editing, ribonucleoprotein (RNP) delivery, and complementation. We also discuss some of the challenges to applying CRISPR-Cas in oomycetes and potential ways to overcome them. Advances in CRISPR-Cas technologies are being used to illuminate the biology of oomycetes, which ultimately can guide the development of tools for managing oomycete diseases.

<i>In vitro</i> regeneration of grape

Considering the global and Russian experience in grape accessions preservation, one of the most reliable ways is the creation of a duplicate in vitro collection. However, in connection with the creation of duplicate grape collections and development of genome editing techniques, there is a need for selecting the most optimal medium composition that will ensure the maximum rate of callus formation and regeneration during the introduction of grape plants into in vitro culture. This will make it possible to obtain the necessary amount of material for further editing and subsequent regeneration of plants with knockout of target genes to improve economically valuable traits. For grapes, this is primarily an increase in resistance to powdery mildew caused by Uncinula necator Burill.The effect of active substances of biological and synthetic origin on the morphogenesis has been studied for many crops, including regenerant plants of the genus Vitis L. However, the genus Vitis is very diverse and heterogeneous in its genetic, physiological and morphological structure, as a result of which the recommended media and components for cultivation under in vitro conditions may not suit every cultivar. Local Russian grape cultivars are better suited to local growing conditions, so efforts should be focused on the development of techniques related to the preservation of local varieties in collections in vitro.Knowledge of genes controlling certain traits, as well as the availability of grape accessions whose genome has been sequenced, contribute to successful in silico analysis for creating editing constructs.

Parthenocarpy-related genes induced by naphthalene acetic acid in oil palm interspecific O × G [Elaeis oleifera (Kunth) Cortés × Elaeis guineensis Jacq.] hybrids.

Parthenocarpy is the development without fertilization of seedless fruits. In the oil palm industry, the development of parthenocarpic fruits is considered an attractive option to increase palm oil production. Previous studies have shown the application of synthetic auxins in Elaeis guineensis, and interspecific O×G hybrids (Elaeis oleifera (Kunth) Cortés × E. guineensis Jacq.) induces parthenocarpy. The aim of this study was to identify the molecular mechanism through transcriptomics and biology system approach to responding to how the application of NAA induces parthenocarpic fruits in oil palm O×G hybrids. The transcriptome changes were studied in three phenological stages (PS) of the inflorescences: i) PS 603, pre-anthesis III, ii) PS 607, anthesis, and iii) PS 700, fertilized female flower. Each PS was treated with NAA, Pollen, and control (any application). The expression profile was studied at three separate times: five minutes (T0), 24 hours (T1), and 48h post-treatment (T2). The RNA sequencing (RNA seq) approach was used with 27 oil palm O×G hybrids for a total of 81 raw samples. RNA-Seq showed around 445,920 genes. Numerous differentially expressed genes (DEGs) were involved in pollination, flowering, seed development, hormone biosynthesis, and signal transduction. The expression of the most relevant transcription factors (TF) families was variable and dependent on the stage and time post-treatment. In general, NAA treatment expressed differentially more genes than Pollen. Indeed, the gene co-expression network of Pollen was built with fewer nodes than the NAA treatment. The transcriptional profiles of Auxin-responsive protein and Gibberellin-regulated genes involved in parthenocarpy phenomena agreed with those previously reported in other species. The expression of 13 DEGs was validated by RT-qPCR analysis. This detailed knowledge about the molecular mechanisms involved in parthenocarpy could be used to facilitate the future development of genome editing techniques that enable the production of parthenocarpic O×G hybrid cultivars without growth regulator application.

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.

The social aspects of genome editing: publics as stakeholders, populations and participants in animal research.

The application of genome editing to animal research connects to a wide variety of policy concerns and public conversations. We suggest focusing narrowly on public opinion of genome editing is to overlook the range of positions from which people are brought into relationships with animal research through these technologies. In this paper, we explore three key roles that publics are playing in the development of genome editing techniques applied to animals in biomedical research. First, publics are positioned by surveys and focus groups as stakeholders with opinions that matter to the development of research technologies. Learning lessons from controversies over genetically modified food in Europe, these methods are used to identify problems in science–society relations that need to be managed. Second, people are recruited into research projects through participating in biobanks and providing data, where their contributions are encouraged by appeals to the public good and maintained by public confidence. Thirdly, patients are increasingly taking positions within research governance, as lay reviewers on funding panels, where their expertise helps align research priorities and practices with public expectations of research. These plural publics do not easily aggregate into a simple or singular public opinion on genome editing. We conclude by suggesting more attention is needed to the multiple roles that different publics expect – and are expected – to play in the future development of genomic technologies.

ALS-associated genes display CNS expression in the developing zebrafish

Amyotrophic lateral sclerosis (ALS) is characterized by progressive muscle atrophy resulting from the deterioration of motor neurons in the central nervous system (CNS). Recent genome-wide association studies have revealed several genes linked to ALS, further demonstrating the complexity of the disease. The zebrafish (Danio rerio) is an attractive model organism to study the function of the rapidly expanding number of ALS-associated genes, in part, due to the development of genome editing techniques that have facilitated specific gene targeting. Before investing in the manipulation and phenotypic examination of these genes, however, it is important to ascertain the localization of expression in this organism. We performed an expression analysis of 29 total ALS-linked genes in the developing zebrafish, specifically focusing on those genes that displayed robust and reproducible expression at multiple different timepoints. First, we classified a subset of the most robustly expressed genes into three distinct groups: head-only expression, head and weak trunk expression, and head and robust trunk expression. Then, we defined the characteristic pattern of each gene at 2, 3, and 4 days post fertilization. This analysis will facilitate improved mutant phenotype assessment in zebrafish by focusing researchers on the areas of expression.

CRISPR-Cas9 mediated genome editing in Caenorhabditis elegans

The development of genome editing techniques based on CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system has revolutionized biomedical researches. It can be utilized to edit genome sequence in almost any organisms including Caenorhabditis elegans, one of the most convenient and classic genetic model animals. The application of CRISPR-Cas9 mediated genome editing in C. elegans promotes the functional analysis of gene and proteins under many physiological conditions. In this mini-review, we summarized the development of CRISPR-Cas9-based genome editing in C. elegans.

Genome Editing of Pig.

Pigs are important livestock for food and have been used in various biomedical studies, particularly translational research, as experimental animals because of their anatomical and physiological similarity to humans. The recent development of genome editing techniques, such as ZFN, TALEN, and CRISPR/Cas9, has rapidly expanded the use of genome editing tools in a variety of animals, resulting in the relatively easy and efficient generation of gene knock-out pigs. In the past few years, there has been a sustained increase in reports describing the development of genetically modified pigs. This chapter introduces our workflow for establishing the genetically modified cells (nuclear donor cells) necessary to create gene knock-out pigs using somatic cell nuclear transfer and focuses on the actual generation of gene knock-out pigs using a cytoplasmic injection method.