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CRISPR/Cas9-mediated Genome-editing Reveals 10 Testis-enriched Genes and One Non-testis-enriched Gene are Dispensable for Male Fecundity in Mice.
BACKGROUND: More than 1000 genes have been identified as predominantly expressed in the human testis. Advances in gene editing technologies have enabled the rapid and efficient generation of genetically engineered mice. This approach facilitates the screening of genes essential for spermatogenesis by analyzing knockout mouse models. OBJECTIVES: This study aimed to elucidate the essential genes in male reproductive function by generating knockout mouse models. MATERIALS AND METHODS: We selected 11 target genes that may have potential roles in the male reproductive system based on a public database. Knockout mouse lines of these target genes were generated using the CRISPR/Cas9 system to elucidate their functions in male reproduction. Also, we conducted natural mating tests to elucidate fecundity and analyzed the phenotype of the knockout males. RESULTS: Natural mating tests revealed that all 11 gene-deficient mouse lines maintained normal male fertility. The phenotypic analysis, including testis appearance and weight, histology of testis and epididymis, and sperm motility and morphology, showed no apparent abnormalities. DISCUSSION AND CONCLUSION: These results suggest that each gene is not essential for male reproductive function.
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Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells.
CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level. This approach enables rapid target discovery and validation in both mouse and human primary T cells. © 2018 by John Wiley & Sons, Inc.
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Biomembrane-inspired lipid nanoparticles enhance CRISPR-Cas9 editing for hemophilia A.
Lipid nanoparticles (LNPs) have substantially advanced RNA-based therapies; however, their use for CRISPR-Cas9 remains limited by sub-optimal endosomal escape, innate immune activation, transient nuclease expression, and restricted tissue specificity. Here, we engineered biomembrane-inspired LNPs containing sphingomyelin and C18-galactosyl ceramide (C18-GalCer) to improve liver-targeted CRISPR delivery. The optimized formulation increased in vivo editing efficiency 2.3-fold relative to a benchmark ALC-0315 LNP. Compared with adeno-associated virus (AAV) vectors, transient mRNA delivery reduced the theoretical risk of insertional mutagenesis, lowered innate immune readouts, and allowed dose titration. By pairing AAV-mediated delivery of a therapeutic Factor VIII donor with LNP-mediated CRISPR-Cas9, we achieved efficient and durable gene correction in a hemophilia A mouse model. A single LNP dose restored plasma Factor VIII activity to >50 % of wild-type levels and maintained this correction for >12 weeks, with low systemic cytokine induction, undetectable off-target insertions, and no overt toxicity. The LNPs retained physicochemical properties and editing potency after prolonged storage and multiple freeze-thaw cycles, supporting their translational potential. Collectively, our findings position biomembrane-inspired LNPs as a safe and efficient non-viral CRISPR platform with potential applicability to other gene-editing therapies.
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Pervasive donor DNA integration defies precision gene editing of hematopoietic stem cells.
Therapeutic gene editing based on homology-directed repair (HDR) promises to precisely recombine donor template sequences at programmed double-strand break sites. In this issue, Ferrari et al. report that commonly used AAV donors blunt hematopoietic repopulation and result in frequent genomic integrations that could interfere with normal gene expression.
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Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.
A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.
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CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation.
CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity of Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, the changes to the DNA sequence that are mediated by CRISPR/Cas9 affect the structures and stability of the genome, which may affect the accuracy of results. Mutations in the RuvC and HNH regions of the Cas9 protein lead to the inactivation of Cas9 into dCas9 with no endonuclease activity. Despite the loss of endonuclease activity, dCas9 can still bind the DNA strand using guide RNA. Recently, proteins with active/inhibitory effects have been linked to the end of the dCas9 protein to form fusion proteins with transcriptional active/inhibitory effects, named CRISPRa and CRISPRi, respectively. These CRISPR tools mediate the transcription activity of protein-coding and non-coding genes by regulating the chromosomal modification states of target gene promoters, enhancers, and other functional elements. Here, we highlight the epigenetic mechanisms and applications of the common CRISPR/dCas9 tools, by which we hope to provide a reference for future related gene regulation, gene function, high-throughput target gene screening, and disease treatment.
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Common strategies in silkworm disease resistance breeding research.
The silkworm, which is considered a model invertebrate organism, was the first insect used for silk production in human history and has been utilized extensively throughout its domestication. However, sericulture has been plagued by various pathogens that have caused significant economic losses. To enhance the resistance of a host to its pathogens,numerous strategies have been developed. For instance, gene-editing techniques have been applied to a wide range of organisms, effectively solving a variety of experimental problems. This review focuses on several common silkworm pests and their pathogenic mechanisms, with a particular emphasis on breeding for disease resistance to control multiple types of silkworm diseases. The review also compares the advantages and disadvantages of transgenic technology and gene-editing systems. Finally, the paper provides a brief summary of current strategies used in breeding silkworm disease resistance, along with a discussion of the establishment of existing technologies and their future application prospects. © 2023 Society of Chemical Industry.
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Potential of Gene Editing and Induced Pluripotent Stem Cells (iPSCs) in Treatment of Retinal Diseases.
The advent of gene editing has introduced the ability to make changes to the genome of cells, thus allowing for correction of genetic mutations in patients with monogenic diseases. Retinal diseases are particularly suitable for the application of this new technology because many retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA), are monogenic. Moreover, gene delivery techniques such as the use of adeno-associated virus (AAV) vectors have been optimized for intraocular use, and phase III trials are well underway to treat LCA, a severe form of inherited retinal degeneration, with gene therapy. This review focuses on the use of gene editing techniques and another relatively recent advent, induced pluripotent stem cells (iPSCs), and their potential for the study and treatment of retinal disease. Investment in these technologies, including overcoming challenges such as off-target mutations and low transplanted cell integration, may allow for future treatment of many debilitating inherited retinal diseases.
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Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model.
The prokaryotic CRISPR/Cas9 system has recently emerged as a powerful tool for genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this genome editing machinery and indeed the very feasibility of using these techniques in vivo remain challenging for most tissue types. Here, we show that nonreplicable Cas9/sgRNA ribonucleoproteins can be used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. We developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatal mice. This method results in rapid gene editing in epidermal stem cells. Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mouse model, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment. This study provides an in vivo gene correction strategy using ribonucleoproteins as curative treatment for genetic diseases in skin and potentially in other somatic tissues.
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Identification of a Rice Leaf Width Gene Narrow Leaf 22 (NAL22) through Genome-Wide Association Study and Gene Editing Technology.
Rice leaf width (RLW) is a crucial determinant of photosynthetic area. Despite the discovery of several genes controlling RLW, the underlying genetic architecture remains unclear. In order to better understand RLW, this study conducted a genome-wide association study (GWAS) on 351 accessions from the rice diversity population II (RDP-II). The results revealed 12 loci associated with leaf width (LALW). In LALW4, we identified one gene, Narrow Leaf 22 (NAL22), whose polymorphisms and expression levels were associated with RLW variation. Knocking out this gene in Zhonghua11, using CRISPR/Cas9 gene editing technology, resulted in a short and narrow leaf phenotype. However, seed width remained unchanged. Additionally, we discovered that the vein width and expression levels of genes associated with cell division were suppressed in nal22 mutants. Gibberellin (GA) was also found to negatively regulate NAL22 expression and impact RLW. In summary, we dissected the genetic architecture of RLW and identified a gene, NAL22, which provides new loci for further RLW studies and a target gene for leaf shape design in modern rice breeding.
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