The Trypanosoma cruzi pleiotropic protein P21 orchestrates the intracellular retention and in-vivo parasitism control of virulent Y strain parasites.

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

CRISPR-Cas9 Protocol for Efficient Gene Knockout and Transgene-free Plant Generation.

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery. Key features • Two sgRNAs employed. • Takes 6-12 months to have an edited transgene-free plant. • Setup in tomato.

CDK5 Deficiency Does not Impair Neuronal Differentiation of Human Induced Pluripotent Stem Cells but Affects Neurite Outgrowth.

Cyclin-dependent kinase 5 (CDK5) is a protein kinase involved in neuronal homeostasis and development critical for neuronal survival. Besides, its deregulation is linked to neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases. For that reason, we aimed to generate a deficient CDK5 genetic model in neurons derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 technology. We obtained a heterozygous CDK5+/- clone for the FN2.1 hiPSC line that retained hiPSC stemness and pluripotent potential. Then, neural stem cells (NSCs) and further neurons were derived from the CDK5+/- KO FN2.1 hiPSCs, and their phenotype was validated by immunofluorescence staining using antibodies that recognize lineage-specific markers (SOX-1, SOX-2, and NESTIN for NSCs and TUJ-1, MAP-5, and MAP-2 for neurons). We found that the proliferation rate increased in CDK5+/- KO hiPSC-derived neurons concomitantly with a reduction in NEUN and P35 expression levels. However, the morphometric analysis revealed that CDK5 deficiency caused an increase in the length of the main, primary, and secondary neurites and the neuronal soma area. As a whole, we found that a deficit in CDK5 does not impair hiPSC neuronal differentiation but deregulates proliferation and neurite outgrowth, favoring elongation. The misregulated activity of specific kinases leads to abnormalities such as impaired axonal connectivity in neurodegenerative diseases. Thus, therapeutic approaches aimed at normalizing the activity of kinases, such as CDK5, may help prevent the degeneration of vulnerable neurons.

Molecular tools to regulate gene expression in Trypanosoma cruzi.

Developing molecular strategies to manipulate gene expression in trypanosomatids is challenging, particularly with respect to the unique gene expression mechanisms adopted by these unicellular parasites, such as polycistronic mRNA transcription and multi-gene families. In the case of Trypanosoma cruzi (T. cruzi), the causative agent of Chagas Disease, the lack of RNA interference machinery further complicated functional genetic studies important for understanding parasitic biology and developing biomarkers and potential therapeutic targets. Therefore, alternative methods of performing knockout and/or endogenous labelling experiments were developed to identify and understand the function of proteins for survival and interaction with the host. In this review, we present the main tools for the genetic manipulation of T. cruzi, focusing on the Clustered Regularly Interspaced Short Palindromic Repeats Cas9-associated system technique widely used in this organism. Moreover, we highlight the importance of using these tools to elucidate the function of uncharacterized and glycosylated proteins. Further developments of these technologies will allow the identification of new biomarkers, therapeutic targets and potential vaccines against Chagas disease with greater efficiency and speed.

Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks.

BACKGROUND: The selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated. RESULTS: We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. CONCLUSIONS: Our study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Droplets for Gene Editing Using CRISPR-Cas9 and Clonal Selection Improvement Using Hydrogels.

Gene editing tools have triggered a revolutionary transformation in the realms of cellular and molecular physiology, serving as a fundamental cornerstone for the evolution of disease models and assays in cell culture reactions, marked by various enhancements. Concurrently, microfluidics has emerged over recent decades as a versatile technology capable of elevating performance and reducing costs in daily experiments across diverse scientific disciplines, with a pronounced impact on cell biology. The amalgamation of these groundbreaking techniques holds the potential to amplify the generation of stable cell lines and the production of extracellular matrix hydrogels. These hydrogels, assuming a pivotal role in isolating cells at the single-cell level, facilitate a myriad of analyses. This study presents a novel method that seamlessly integrates CRISPR-Cas9 gene editing techniques with single-cell isolation methods in induced pluripotent stem cell (hiPSC) lines, utilizing the combined power of droplets and hydrogels. This innovative approach is designed to optimize clonal selection, thereby concurrently reducing costs and the time required for generating a stable genetically modified cell line. By bridging the advancements in gene editing and microfluidic technologies, our approach not only holds significant promise for the development of disease models and assays but also addresses the crucial need for efficient single-cell isolation. This integration contributes to streamlining processes, making it a transformative method with implications for enhancing the efficiency and cost-effectiveness of stable cell line generation. As we navigate the intersection of gene editing and microfluidics, our study marks a significant stride toward innovative methodologies in the dynamic landscape of cellular and molecular physiology research.

Novelty in improvement of CAR T cell-based immunotherapy with the aid of CRISPR system

Abstract Introduction Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. Method In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. Results The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. Conclusion: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus.

Fungi belonging to the genus Pseudogymnoascus have garnered increasing attention in recent years. One of the members of the genus, P. destructans, has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of Pseudogymnoascus species has expanded, in parallel with the increased availability of genome sequences. Moreover, Pseudogymnoascus exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of Pseudogymnoascus remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in Pseudogymnoascus, using an Antarctic strain of Pseudogymnoascus verrucosus as a model. Both methods were applied to target azpA, a gene involved in red pigment biosynthesis. Silencing of the azpA gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating azpA and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in Pseudogymnoascus research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.

Novelty in improvement of CAR T cell-based immunotherapy with the aid of CRISPR system.

INTRODUCTION: Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. METHOD: In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. RESULTS: The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. CONCLUSION: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

Genetic variations in tumor-suppressor miRNA-encoding genes and their target genes: focus on breast cancer development and possible therapeutic strategies.

MicroRNAs (miRNAs) negatively affect gene expression by binding to their specific mRNAs resulting in either mRNA destruction or translational repression. The aberrant expression of various miRNAs has been associated with a number of human cancer. Oncogenic or tumor-suppressor miRNAs regulate a variety of pathways involved in the development of breast cancer (BC), including cell proliferation, apoptosis, metastasis, cancer recurrence, and chemoresistance. Variations in miRNA-encoding genes and their target genes lead to dysregulated gene expression resulting in the development and progression of BC. The various therapeutic approaches to treat the disease include chemotherapy, radiation therapy, surgical removal, hormone therapy, chemotherapy, and targeted biological therapy. The purpose of the current review is to explore the genetic variations in tumor-suppressor miRNA-encoding genes and their target genes in association with the disease development and prognosis. The therapeutic interventions targeting the variants for better disease outcomes have also been discussed.

Identification of early flowering mutant gene in Phalaenopsis amabilis (L) Blume for sgRNA construction in CRISPR/Cas9 genome editing system / Identificação do gene mutante de floração precoce em Phalaenopsis amabilis (L) Blume para construção de sgRNA no sistema de edição de genoma CRISPR/Cas9

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented.

Phalaenopsis amabilis (L.) Blume, comumente chamada de orquídea mariposa (Orchidaceae), é uma espécie natural de orquídea designada como a flor nacional da Indonésia por seu belo formato de flor e período de floração duradouro. Basicamente, P. amabilis tem uma longa fase vegetativa que causa floração tardia, cerca de 2 a 3 anos para a floração, portanto, um método para encurtar o período vegetativo é desejado. A mais recente abordagem tecnológica que pode ser utilizada para acelerar a floração de P. amabilis é o método de edição do genoma CRISPR/Cas9 para inativar o GAI (Gibberellic Acid Insensitive) que pode ser usado como um gene mutante para acelerar a regulação da floração dos genes FLOWERING TIME (FT), via de biossíntese. Para isto, a melhor abordagem é silenciar o GAI gene com um sistema knockout que deve ser iniciado com a identificação e caracterização do gene alvo GAI no P. amabilis, e que, posteriormente, será utilizado como um único RNA guia. A eficiência de nocaute mediada por CRISPR/Cas9 é altamente dependente das propriedades do sgRNA usados. O SgRNA consiste em uma sequência alvo, determinando seu desempenho de especificidade. Executamos agrupamento filogenético para a proteína PaGAI com espécies de orquídeas intimamente relacionadas, como Dendrobium capra, Dendrobium cultivars e Cymbidium sinensis. SWISS-Model foi utilizado como ferramenta webserver para modelagem de homologia de estruturas de proteínas. Os resultados mostram que P. amabilis possui um domínio específico com ocorrência de mutações pontuais nos dois domínios conservativos. Portanto, uma única reconstrução de RNA guia precisa ser implementada.

Novel approaches for HTLV-1 therapy: innovative applications of CRISPR-Cas9

ABSTRACT The human T-cell lymphotropic virus type 1 (HTLV-1) is a single-stranded positive-sense RNA virus that belongs to the Retroviridae family, genus Deltaretro, and infects approximately five to 10 million people worldwide. Although a significant number of individuals living with HTLV-1 remain asymptomatic throughout their lives, some develop one or more severe clinical conditions, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin's lymphoma with a more threatening course known as adult T-cell leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited and focus primarily on treating symptoms and controlling viral latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the intricate links associated with HTLV-1. By targeting or silencing key genes during initial infection and dysregulating immune signaling pathways, CRISPR-Cas9 offers potential intervention opportunities. In this review, we address the therapeutic potential of CRISPR-Cas9 gene editing, as well as examine the primary mechanisms involved in editing potential target genes and discuss the existing evidence in the current scientific literature.

An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.

Breast cancer is one of the leading causes of death in women around the world. Over time, many genes and mutations that are associated with the development of this disease have been identified. However, the specific role of many genes has not yet been fully elucidated. Higher ARID4B expression has been identified as a risk factor for diverse cancer types. Silencing experiments also showed that ARID4B is associated with developing cancer-associated characteristics. However, no transcriptomic studies have shown the overall cellular effect of loss of function in breast cancer in humans. This study addresses the impact of loss-of-function mutations in breast cancer MCF-7 cells. Using the CRISPR/Cas9 system, we generated mutations that caused heterozygous truncated proteins, isolating three monoclonal lines carrying insertions and deletions in ARID4B. We observed reduced proliferation and migration in in vitro experiments. In addition, from RNA-seq assays, a differential expression analysis shows known and novel deregulated cancer-associate pathways in mutated cells supporting the impact of ARID4B. For example, we found the AKT-PI3K pathway to be altered at the transcript level but through different genes than those reported for ARID4B. Our transcriptomic results also suggest new insights into the role of ARID4B in aggressiveness by the epithelial-to-mesenchymal transition and TGF-ß pathways and in metabolism through cholesterol and mevalonate pathways. We also performed exome sequencing to show that no off-target effects were apparent. In conclusion, the ARID4B gene is associated with some aggressive phenotypes in breast cancer cells.

gRNA Design: How Its Evolution Impacted on CRISPR/Cas9 Systems Refinement.

Over the past decade, genetic engineering has witnessed a revolution with the emergence of a relatively new genetic editing tool based on RNA-guided nucleases: the CRISPR/Cas9 system. Since the first report in 1987 and characterization in 2007 as a bacterial defense mechanism, this system has garnered immense interest and research attention. CRISPR systems provide immunity to bacteria against invading genetic material; however, with specific modifications in sequence and structure, it becomes a precise editing system capable of modifying the genomes of a wide range of organisms. The refinement of these modifications encompasses diverse approaches, including the development of more accurate nucleases, understanding of the cellular context and epigenetic conditions, and the re-designing guide RNAs (gRNAs). Considering the critical importance of the correct performance of CRISPR/Cas9 systems, our scope will emphasize the latter approach. Hence, we present an overview of the past and the most recent guide RNA web-based design tools, highlighting the evolution of their computational architecture and gRNA characteristics over the years. Our study explains computational approaches that use machine learning techniques, neural networks, and gRNA/target interactions data to enable predictions and classifications. This review could open the door to a dynamic community that uses up-to-date algorithms to optimize and create promising gRNAs, suitable for modern CRISPR/Cas9 engineering.

Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol.

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

Therapeutic applications of CRISPR/Cas9 mediated targeted gene editing in acute lymphoblastic leukemia: current perspectives, future challenges, and clinical implications.

Acute Lymphoblastic Leukemia (ALL) is the predominant hematological malignancy in pediatric populations, originating from B- or T-cell precursors within the bone marrow. The disease exhibits a high degree of heterogeneity, both at the molecular level and in terms of clinical presentation. A complex interplay between inherited and acquired genetic alterations contributes to disease pathogenesis, often resulting in the disruption of cellular functions integral to the leukemogenic process. The advent of CRISPR/Cas9 as a gene editing tool has revolutionized biological research, underscoring its potential to modify specific genomic loci implicated in cancer. Enhanced understanding of molecular alterations in ALL has facilitated significant advancements in therapeutic strategies. In this review, we scrutinize the application of CRISPR/Cas9 as a tool for identifying genetic targets to improve therapy, circumvent drug resistance, and facilitate CAR-T cell-based immunotherapy. Additionally, we discuss the challenges and future prospects of CRISPR/Cas9 applications in ALL.