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Evolution of Prime Editing: Enhancing Efficiency and Expanding Capacity.
Genetic mutations cause approximately 80% of rare human diseases, highlighting the urgent need for precise genome editing. Since clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) nucleases were first used for genome editing in 2012, genome editing technologies have rapidly advanced. Base editors, derived from the CRISPR-Cas system, were developed to introduce specific point mutations without requiring DNA double-strand breaks, and subsequently, prime editing (PE) technology was created to enable insertions, deletions, and all types of point mutations. The precision and versatility of PE make it a promising tool for clinical applications. However, PE has potential limitations, including low editing efficiency and limited capacity for large-scale manipulation. To overcome these limitations, research has been continuously conducted to improve PE efficiency and expand its capabilities. Therefore, this review aims to highlight current efforts and future directions for developing and improving PE-related tools.
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Sequence modification on demand: search and replace tools for precise gene editing in plants.
Until recently, our ability to generate allelic diversity in plants was limited to introduction of variants from domesticated and wild species by breeding via uncontrolled recombination or the use of chemical and physical mutagens-processes that are lengthy and costly or lack specificity, respectively. Gene editing provides a faster and more precise way to create new variation, although its application in plants has been dominated by the creation of short insertion and deletion mutations leading to loss of gene function, mostly due to the dependence of editing outcomes on DNA repair pathway choices intrinsic to higher eukaryotes. Other types of edits such as point mutations and precise and pre-designed targeted sequence insertions have rarely been implemented, despite providing means to modulate the expression of target genes or to engineer the function and stability of their protein products. Several advancements have been developed in recent years to facilitate custom editing by regulation of repair pathway choices or by taking advantage of alternative types of DNA repair. We have seen the advent of novel gene editing tools that are independent of DNA double-strand break repair, and methods completely independent of host DNA repair processes are being increasingly explored. With the aim to provide a comprehensive review of the state-of-the-art methodology for allele replacement in plants, I discuss the adoption of these improvements for plant genome engineering.
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CRISPR-derived genome editing technologies for metabolic engineering.
In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.
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Anthraquinone from Edible Fungi Pleurotus ostreatus Protects Human SH-SY5Y Neuroblastoma Cells Against 6-Hydroxydopamine-Induced Cell Death-Preclinical Validation of Gene Knockout Possibilities of PARK7, PINK1, and SNCA1 Using CRISPR SpCas9.
Parkinson's disease (PD) results from the degeneration of the nervous tissue brought about by ecological and hereditary components which affects nerve cells in the brain. It is the world's second most normal neurodegenerative issue, which can essentially weaken the personal satisfaction, make reliance, and trigger untimely mortality of affected people. The commonness pace of PD is 0.5-1% among individuals in the age group of 65-69 years and 1-3% among those 80 or more. Clinical appearances incorporate bradykinesia, tremors, unbending nature, and postural unsteadiness; spectrums of non-motor symptoms include psychological hindrance and passionate and behavioral brokenness. In this study, 6-OHDA-induced neurotoxicity was analyzed for various cytotoxicity analyses. The genes identified were PINK1 (PTEN-induced kinase 1), PARK7 (Parkinsonism-associated deglycase) and SNCA 1 (alpha synuclein1) validated using CRISPR spcas9 genome editing tool. In this study, Anthraquinone isolated from Pleurotus ostreatus was treated against a dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), which induced neurotoxicity in SH-SY5Y cells. Experimental groups in SH-SY5Y neuroblastoma cells were treated with anthraquinone (50 nM) and 6-OHDA (100 nM). MTT and ROS assays were performed to assess the cell viability and oxidative stress within the cells, followed by mixed-member proportional (Mitochondrial membrane potential), dual staining, and immunoblotting. 6-OHDA-induced cell death in SH-SY5Y cells was dose-dependently attenuated by treatment with anthraquinone. The genes responsible for mutation were studied and the mutated RNAs knockout possibilities was studied using CRISPR spcas9 genome editing tool. Treatment with anthraquinone attenuated the level of oxidative stress and reduced the mitochondrial dysfunction associated with 6-OHDA treatment. Immunoblot analysis carried out with apoptotic markers showed that cytochrome C and caspase-3 expression increased significantly in anthraquinone-treated cells, whereas 6-OHDA-treated group showed a significant decrease when compared with an experimental control group. The mutated genes PARK7, PINK1, and SNCA1 were analyzed and found to exhibit four gene knock possibilities to treat PD. Reports demonstrate that other than following up on the biosynthesis of dopamine and its metabolites, these mixes counteract D2 receptors' extreme touchiness. It is proposed that further examinations need be directed to better understand the activity of the bioactive mixes circulated in these edible fungi Pleurotus ostreatus. The gene knockout possibilities identified by CRISPR SpCas9 will pave a way for better research for PD treatment.
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Editing a gibberellin receptor gene improves yield and nitrogen fixation in soybean.
Soybean is an important source of oil, protein, and feed. However, its yield is far below that of major cereal crops. The green revolution increased the yield of cereal crops partially through high-density planting of lodging-resistant semi-dwarf varieties, but required more nitrogen fertilizers, posing an environmental threat. Genes that can improve nitrogen use efficiency need to be integrated into semi-dwarf varieties to avoid the overuse of fertilizers without the loss of dwarfism. Unlike cereal crops, soybean can assimilate atmospheric nitrogen through symbiotic bacteria. Here, we created new alleles of GmGID1-2 (Glycine max GIBBERELLIN INSENSITIVE DWARF 1-2) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) editing, which improved soybean architecture, yield, seed oil content, and nitrogen fixation, by regulation of important pathways and known genes related to branching, lipid metabolism, and nodule symbiosis. GmGID1-2 knockout reduced plant height, and increased stem diameter and strength, number of branches, nodes on the primary stem, pods, and seeds per plant, leading to an increase in seed weight per plant and yield in soybean. The nodule number, nodule weight, nitrogenase activity, and nitrogen content were also improved in GmGID1-2 knockout soybean lines, which is novel compared with the semi-dwarf genes in cereal crops. No loss-of-function allele for GmGID1-2 was identified in soybean germplasm and the edited GmGID1-2s are superior to the natural alleles, suggesting the GmGID1-2 knockout mutants generated in this study are valuable genetic resources to further improve soybean yield and seed oil content in future breeding programs. This study illustrates the pleiotropic functions of the GID1 knockout alleles with positive effects on plant architecture, yield, and nitrogen fixation in soybean, which provides a promising strategy toward sustainable agriculture.
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Mimicking natural polymorphism in eIF4E by CRISPR-Cas9 base editing is associated with resistance to potyviruses.
In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus-resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss-of-function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR-nCas9-cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single-point mutation through C-to-G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome-editing technologies offers a feasible solution for engineering transgene-free genetic resistance in plants, even across species barriers.
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Towards CRISPR-based editing of the mitochondrial genome in yeast.
Mitochondria, which evolved from symbiotic bacteria, possess their own genomes (mtDNA) and support independent transcription and translation within the organelle. Given the essential role of mtDNA in energy production, metabolism, as well as cellular homeostasis, and the high density of confirmed pathogenic mutations that map to mtDNA, there is a pressing need for versatile methods to study and manipulate this genome. Although CRISPR technology has revolutionized the editing of nuclear genomes, it has not been successfully extended to mtDNA, primarily due to the challenge of delivering single guide RNAs (sgRNAs) across both outer and inner mitochondrial membranes. Here we develop a survival-based reporter in Saccharomyces cerevisiae to screen for potential RNA import motifs. We identify a 40-nucleotide aptamer (IM83) that facilitates sgRNA entry into the mitochondrial matrix, enabling CRISPR editing by a mitochondrially-localized adenine base editor. We show that mitochondrial import of IM83 is ATP-dependent and enhanced by the tRNA synthetase Msk1. Further investigations identify barriers to efficient CRISPR editing of mtDNA, including loss of membrane potential associated with mitochondrial targeting of the base editor. These insights lay the groundwork for future improvements in CRISPR-based editing of mtDNA in eukaryotes.
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A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant.
The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs in vitro, followed by assessment in c.1222C>T humanized mice in vivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.
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Genome Editing Approaches in Flax (Linum usitatissimum L.): From Tools to Trait Improvement.
Genome editing, particularly CRISPR/Cas-based systems, has emerged as a key tool for functional genomics and trait improvement in flax (Linum usitatissimum L.), an important fiber and oilseed crop. This review focuses specifically on flax as an emerging target species and distinguishes experimentally validated applications from approaches adapted from model plants. Recent progress includes the characterization of endogenous U6 promoters, which improved guide RNA expression and contributed to enhanced genome editing performance under optimized conditions. Reported studies demonstrate efficient targeted mutagenesis in flax; however, editing outcomes remain strongly dependent on genotype, construct design, and regeneration capacity, and stable homozygous edited lines are still limited. Target genes include pathways involved in lignin and cellulose biosynthesis, fatty acid metabolism, and stress responses, influencing fiber quality, oil composition, and stress adaptation. Despite current bottlenecks such as low homologous recombination efficiency and regeneration constraints, base editing, prime editing, and multiplex CRISPR systems provide promising avenues for precision breeding in flax.
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Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid Wheat.
Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.
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