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CRISPR-Cas9 genome editing in Corallochytrium limacisporum,a key species for understanding animal origins.
Microbial holozoans are the closest unicellular relatives of animals. They share a substantial gene repertoire with animals and exhibit complex life cycles. Studying these organisms is crucial for understanding the evolution of multicellularity, and significant progress has been made in uncovering key aspects of the biology of the four microbial holozoans lineages: choanoflagellates, filastereans, ichthyosporeans and corallochytreans. However, reverse genetic tools are still lacking in corallochytreans, one of the earliest-branching holozoan lineages and the only known group with both coenocytic and binary fission development. Here, we present CRISPR-Cas9-mediated gene inactivation and point mutation methodologies in the corallochytrean Corallochytrium limacisporum. As a proof of concept, we inactivated the fkb12 gene, a component of the mTOR pathway, conferring rapamycin resistance, and introduced a point mutation in sdhB, encoding a subunit of succinate dehydrogenase, conferring carboxin resistance. Our results demonstrate the presence of both non-homologous end-joining and homology-directed repair pathways in C. limacisporum and shows an editing efficiency of approximately 2%. Furthermore, simultaneous gene targeting revealed a co-editing frequency of approximately 20%. Finally, this study establishes unequivocally that C. limacisporum is haploid, making it an ideal model for genetic studies and gene editing applications to unravel the molecular mechanisms involved in animal origins.
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High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.
UNLABELLED: Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. STATEMENT OF SIGNIFICANCE: CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type.
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Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics.
Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.
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Gene Edited Tumor Cells Kill and Prevent Cancer in Mice.
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[CRISPR/Cas9 and gene therapy].
The development of techniques that allow the precise and efficient edition of the genome of living cells is one of the main goals of biomedical research. Over the last few decades, a number of genome editing tools have been developed, the most prominent being the CRISPR/Cas9 system, a bacterial defense mechanism that has been redesigned for its use in other cellular systems. The accessibility, both technical and economical, and the enormous potential of CRISPR/Cas9 have contributed to an almost unprecedented revolution in the biomedical sciences and represent an important step forward in the field of gene therapy that needs, however, to be taken cautiously.
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A method for generating genome edited plant lines from CRISPR-transformed Shanxin poplar plants.
Due to the reason of low efficiency of mutation in CRISPR-editing, a high frequency of CRISPR transformed plant lines failing in mutation had been generated and had to be discarded. In the present study, we built a method to increase the efficiency of CRISPR-editing. We used Shanxin poplar (Populus davidiana×P. bolleana) as the study material, and CRISPR-editing system was first built to generate the CRISPR-transformed lines. The line that failed in CRISPR-editing was used for improving the efficiency of mutation, which was treated with heat (37 °C) to improve the cleaving activity of Cas9, leading to increased frequency of the cleaved DNA. Our results indicated that 87-100% of cells in CRISPR-transformed plants whose DNA had been cleaved by heat treatment, and the heat treatment plants were then cut into explants to differentiate adventitious buds. Each differentiated bud can be considered as an independent line. Twenty independent lines were randomly selected for analysis, and all of them had been mutated by CRISPR editing, displaying 4 types of mutation. Our results indicated that heat treatment combined with re-differentiation can generate CRISPR-edited plants efficiently. This method could conquer the problem of low mutation efficiency of CRISPR-editing in Shanxin poplar, and will have a wide application in plant CRISPR-editing.
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Considerations on genetic engineering: regarding the birth of twins subjected to gene edition.
In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.
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A Nucleic Acid/Gold Nanorod-Based Nanoplatform for Targeted Gene Editing and Combined Tumor Therapy.
The CRISPR/Cas9 gene-editing system has become a promising strategy for tumor therapy with its powerful oncogene-editing ability. However, the efficient delivery of sgRNA/Cas9 complex into target tumor cells remains a challenge. Herein, we report a facile strategy for the construction of an sgRNA/Cas9 complex co-assembled nanoplatform for targeted gene editing and combined tumor therapy. In our design, the TAT peptide and thiolated DNA linker functionalized gold nanorod can efficiently load the sgRNA/Cas9 complex through the hybridization between the 3' overhang of sgRNA and the DNA linker. Due to the integration of an active cell targeting group (aptamer) and nuclear targeting peptide (TAT), the multifunctional nanoplatform can elicit the targeted cellular internalization and efficient nuclear targeting transportation to realize endogenous RNase H activated gene editing of the tumor-associated gene polo-like kinase 1 (PLK1). With mild photothermal treatment, this sgRNA/Cas9 complex loaded nanoplatform achieved efficient inhibition of tumor cell proliferation. This multifunctional nanocarrier provides a new strategy for the development of combined tumor therapy.
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An Overview of Programmable Epigenetic Editing Based on CRISPR Tools for Gene Expression Regulation.
Epigenetic regulation provides a dynamic and reversible layer of gene control that functions independently of changes in DNA sequence, primarily mediated by DNA methylation, histone modifications, and higher-order chromatin organization. Aberrant epigenetic states contribute to a wide range of human diseases. However, conventional epigenetic therapies based on small-molecule inhibitors lack locus specificity and often cause global chromatin disturbances. The emergence of programmable epigenetic editing technologies has transformed the field by enabling targeted rewriting of chromatin states at defined genomic loci. Catalytically inactive CRISPR/Cas9 platforms fused to transcriptional activators, repressors, or chromatin-modifying enzymes now allow precise addition or removal of epigenetic marks without altering the underlying DNA sequence. This chapter provides an overview of the conceptual and technical foundations of CRISPR-based epigenetic editing, including tools for gene activation and repression, DNA methylation, histone modifications, and multiplexed systems that permit coordinated regulation of multiple genomic loci or epigenetic marks. Delivery methods for in vitro and in vivo applications are discussed, with an emphasis on viral and nonviral platforms that enable tissue-specific, durable gene regulation. Finally, recent preclinical and clinical studies highlight the potential of programmable epigenetic editing as a next-generation therapy for precise and reversible gene control.
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Abnormal expression pattern of knock-in marker in Eimeria tenella using CRISPR/Cas9.
Gene editing technology has been widely applied in the genetic manipulation of many organisms and is increasingly being utilized in eukaryotic pathogens. However, its efficiency often requires improvement. In our study using CRISPR/Cas9 to genetically manipulate Eimeria tenella, we aimed to insert a tag into a target gene locus via homologous recombination, but observed outcomes inconsistent with expectations. Whole-genome sequencing analysis of the integration sites revealed that the transgenic E. tenella did not exhibit correct targeted integration. These results indicate that creating double-strand breaks (DSB) at specific genomic sites to trigger homology-directed repair (HDR) for gene modification can lead to mislocalized expression. This study provides insights for utilizing CRISPR/Cas9 technology in genetic editing, particularly in E. tenella, and offers suggestions for improving strategies that employ the co-transfection of multiple plasmids, such as Cas9-gRNA and donor plasmids.
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