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Towards a novel therapy against AIDS.
AIDS is an infectious disease that kills over a million people per year. Very recently, Dash et al have for the first time reached the functional cure in HIV-infected humanized mice using CRISPR-Cas9 in combination with LASER ART, and this with a success of one third. Here, I use a theoretical approach to design a therapeutic strategy applicable to humans and different from that of Dash et al. The experimental treatment presented here includes the injection of an Env-directed integrase-defective CRISPR gene-editing lentiviral vector able to express quintuplex gRNAs plus the humanized SpCas9 and the puromycin resistance gene linked by T2A, preceded by a plasma/leukapheresis and the injection of an immunosuppressive cocktail, and followed by an in vivo positive selection. My protocol could have a major impact on HIV-infected people in the event of confirmation by a clinical trial, and it is possible that it becomes a reference treatment against AIDS, although, for the moment, it is only at the stage of hypothesis and theory.
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A novel fluorescent surfactant enhances the delivery of the Cas9 ribonucleoprotein and enables the identification of edited cells.
In this report, we designed and synthesized a novel fluorescent single tailed surfactant (termed FEDS), which can disrupt endosomes, complex lipofectamine, and can also identify cells that have been transfected. FEDS was able to increase the gene editing efficiency of lipofectamine/Cas9 ribonucleoprotein by 300% via a combination of fluorescent based enrichment and endosomal disruption.
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Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 Components for Genome Editing.
Lipid nanoparticles (LNPs) are a clinically validated nonviral platform for the delivery of CRISPR-associated components. Composed of ionizable lipids, phospholipids, cholesterol, and PEG-lipids, LNPs enable the efficient encapsulation, protection, and cytosolic delivery of therapeutic cargo such as DNA, RNA, or proteins. The clinical relevance of LNPs has already been shown by multiple FDA-approved therapies, including siRNA-based treatments and mRNA vaccines. Compared with viral vectors, LNPs offer several advantages, including reduced immunogenicity, absence of genomic integration, scalable manufacturing, and flexibility in cargo size, while supporting transient expression, which is desirable for genome editing applications. However, challenges remain, including limited tissue specificity and inefficient endosomal escape. Recent advances in lipid chemistry optimization and surface modification have improved delivery performance. Among available formulation techniques, microfluidic mixing has emerged as a preferred method due to its reproducibility, scalability, and precise control over particle properties. This protocol describes a standardized microfluidic workflow for reproducible LNP formulation, providing practical guidance on lipid preparation, nanoparticle assembly, and quality control.
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Protocol for CRISPR-Cas9-mediated genome editing to study spermatogenesis in Caenorhabditis elegans.
Gene silencing by P-element-induced wimpy testis-interacting RNAs is a mechanism to maintain genome integrity in germ cells. Here, we present a protocol for knockin or knockout editing of male germline genome mediated by CRISPR-Cas9 technology in Caenorhabditis elegans. We describe steps for constructing edited plasmids, microinjecting worms with these plasmids, and screening edited worms. We then detail procedures for dissecting released sperm and their observation with fluorescence microscopy. Engineered worms provide a model for studying hermaphrodite/male fertility or protein localization in vivo. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).1.
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Genome Editing of Medaka.
Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knock-out strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knock-in using a plasmid carrying a sequence that does not cause off-target effects in medaka.
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CRISPR-Cas9 mediated proteinase 3 autoantigen deletion as a treatment strategy for anti-neutrophil cytoplasmic autoantibody-associated vasculitis.
INTRODUCTION: Proteinase 3 (PR3) is a major autoantigen in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Here, we performed a proof-of-principle study using ex vivo CRISPR-Cas9 guided gene editing to eliminate the PR3 autoantigen as an alternative to suppressing the autoimmune response to PR3. METHODS: A ribonucleoprotein (RNP) complex of Cas9 protein and a PR3-specific single guide-RNA was transfected into human CD34+ hematopoietic stem and progenitor cells (HSPC) by electroporation. Effects on PR3 protein abundance, neutrophil differentiation, and ANCA-dependent and -independent neutrophil responses were assessed. RESULTS: Gene editing introduced a frame shift in exon 2 of PRTN3. Consequently, PR3 protein was efficiently reduced in neutrophil-differentiated HSPCs as demonstrated by immunoblotting, ELISA, microscopy, and the complete absence of PR3-specific proteolytic activity. Human neutrophil elastase served as control and was not affected. PR3-deleted (PR3KO)- and PR3 wild-type (PR3WT)-HSPCs showed similar neutrophil differentiation. Importantly, general neutrophil defense functions to non-ANCA receptor-independent and -dependent stimuli were similar in PR3KO- and PR3WT-neutrophils as was constitutive apoptosis. Flow cytometry showed that cell membrane-PR3 was significantly reduced on PR3KO-neutrophils and consequent neutrophil activation to either monoclonal antibodies to PR3 or human PR3-ANCA was attenuated. In contrast, myeloperoxidase-ANCA stimulation was not affected. CONCLUSIONS: We show the feasibility and efficacy of depleting the PR3 autoantigen in human CD34+ HSPCs using CRISPR-Cas9. Depleting the PR3 autoantigen instead of suppressing the autoimmune response to PR3 could potentially lead to drug-free remission, particularly in patients with refractory or relapsing disease.
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CRISPR-Cas9 gene-editing to assess the role of RF-amide-related peptide 3 in ovine seasonal breeding.
Seasonal breeding is an adaptive strategy that ensures the birth of the offspring during the best time of year and allows energy saving in times of food scarcity and adverse environmental conditions. At temperate and polar latitudes, photoperiod is the main synchroniser of seasonal functions, which exerts its action through melatonin. Within the pars tuberalis of the anterior pituitary, melatonin triggers a series of events that lead to enhanced local triiodothyronine (T3) production in the medio-basal hypothalamus specifically under long days and ultimately drive the appropriate GnRH output at the median eminence. How T3 governs GnRH output is mostly unknown but neuronal populations that respectively produce KISS1 and RFRP3 appear to be involved. However, while the role of KISS1 as a major GnRH secretagogue is undisputed, the function of RFRP3 in the control of (seasonal) breeding remains enigmatic, with conflicting results hinting at elusive mechanisms. Therefore, we launched an extensive series of experiments in sheep, aimed at invalidating the NPVF gene, which encodes RFRP3, using CRISPR-Cas9 technology. Here, we report on the generation of six sheep for which the NPVF gene has been successfully edited. Four of these animals bear at least one allele expected to behave as a null and constitute founders for distinct ovine lines. These founder sheep will now enter a standard breeding scheme in order to obtain male and female homozygotes for distinct mutations. These animals are expected to provide a clear delineation of the function of RFRP3 in seasonal breeding.
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An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes.
BACKGROUND: Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies. RESULTS: We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used 'half-seed' explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75-100% mutation efficiencies. Indel frequencies varied widely among plants (3-100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27-100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants. CONCLUSIONS: Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.
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Mechanisms and applications of epigenome editing in plants: current status, challenges and future perspectives.
Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.
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Single-Cell Profiling of Lineages and Cell Types in the Vertebrate Brain.
CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types.
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