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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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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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Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science.
The recent discovery and subsequent development of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease.
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De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.
BACKGROUND: Mutants are important for the discovery of functional genes and creation of germplasm resources. Mutant acquisition depends on the efficiency of mutation technology and screening methods. CRISPR-Cas9 technology is an efficient gene editing technology mainly used for editing a few genes or target sites, which has not been applied for the construction of random mutant libraries and for the de novo discovery of functional genes. RESULTS: In this study, we first sequenced and assembled the chromosome-level genome of wild-type rare minnow (Gobiocypris rarus) as a susceptible model of hemorrhagic disease, obtained a 956.05 Mb genome sequence, assembled the sequence into 25 chromosomes, and annotated 26,861 protein-coding genes. Thereafter, CRISPR-Cas9 technology was applied to randomly mutate the whole genome of rare minnow with the conserved bases (TATAWAW and ATG) of the promoter and coding regions as the target sites. The survival rate of hemorrhagic disease in the rare minnow gradually increased from 0% (the entire wild-type population died after infection) to 38.24% (F3 generation). Finally, 7 susceptible genes were identified via genome comparative analysis and cell-level verification based on the rare minnow genome. CONCLUSIONS: The results provided the genomic resources for wild-type rare minnow, and confirmed that the random mutation system designed using CRISPR-Cas9 technology in this study is simple and efficient and is suitable for the de novo discovery of functional genes and creation of a germplasm resource related to qualitative traits.
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Endonuclease mediated genome editing in drug discovery and development: promises and challenges.
Site specific genome editing has been gradually employed in drug discovery and development process over the past few decades. Recent development of CRISPR technology has significantly accelerated the incorporation of genome editing in the bench side to bedside process. In this review, we summarize examples of applications of genome editing in the drug discovery and development process. We also discuss current hurdles and solutions of genome editing.
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PCdb: A comprehensive plant genome-editing database integrating sgRNA efficiency, off-target predictions, and epigenomic landscapes.
CRISPR-Cas-based genome editing has transformed plant biotechnology by enabling precise genomic modifications for crop improvement and functional genomics. The success of these applications hinges on the design of single guide RNAs (sgRNAs) that maximize on-target efficiency while minimizing off-target effects. However, existing resources for sgRNA design and performance evaluation in plants remain fragmented and lack integration with genomic and epigenomic contexts that influence both editing efficacy and specificity. Here, we present PCdb (Plant CRISPR Database; https://gmo.sjtu.edu.cn/pcdb), a comprehensive plant-focused database that integrates experimentally validated sgRNAs, annotated genomic contexts, genome-wide off-target predictions, and multilayer epigenomic annotations. PCdb encompasses 6172 manually curated editing records from 2132 publications, covering 4320 unique sgRNAs and 6 117 424 predicted off-target sites across nine major plant species. Notably, PCdb contextualizes potential editing outcomes-both on target and off target-within the chromatin landscape by incorporating DNA methylation profiles, chromatin accessibility data, and histone modification patterns. The database features an intuitive web interface that supports flexible queries, interactive visualization tools, and comprehensive analytical modules for sgRNA efficiency assessment and off-target analysis. A case study reanalysis of Oryza sativa yield-related genes demonstrates PCdb's ability to generate a detailed performance profile by evaluating both on-target characteristics and off-target risks within their native epigenomic context. Systematic analysis of the database further identifies key sequence and chromatin features that influence editing outcomes, providing novel insights to improve gene-editing efficacy and specificity.
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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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Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro.
Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5'TG3'-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-β1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.
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Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a.
The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.
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