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Efficient Gene Reframing Therapy for Recessive Dystrophic Epidermolysis Bullosa with CRISPR/Cas9.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination or non-homologous end-joining pathways. The non-homologous end-joining pathway, which is activated more frequently than homologous recombination, is prone to introducing small insertions and/or deletions at the double-strand break site, leading to changes in the reading frame. We hypothesized that the non-homologous end-joining pathway is applicable to genetic diseases caused by a frameshift mutation through restoration of the reading frame. Recessive dystrophic epidermolysis bullosa is a hereditary skin disorder caused by mutations in COL7A1. In this study, we applied gene reframing therapy to a recurrent frameshift mutation, c.5819delC, in COL7A1, which results in a premature termination codon. CRISPR/Cas9 targeting this specific mutation site was delivered to recessive dystrophic epidermolysis bullosa patient fibroblasts. After genotyping a large collection of gene-edited fibroblast clones, we identified a significant number (17/50) of clones in which the frameshift in COL7A1 was restored. The reframed COL7 was functional, as shown by triple-helix formation assay in vitro, and was correctly distributed in the basement membrane zone in mice. Our data suggest that mutation site-specific non-homologous end-joining might be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.
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Mice Against Ticks: an experimental community-guided effort to prevent tick-borne disease by altering the shared environment.
Mice Against Ticks is a community-guided ecological engineering project that aims to prevent tick-borne disease by using CRISPR-based genome editing to heritably immunize the white-footed mice ( Peromyscus leucopus) responsible for infecting many ticks in eastern North America. Introducing antibody-encoding resistance alleles into the local mouse population is anticipated to disrupt the disease transmission cycle for decades. Technology development is shaped by engagement with community members and visitors to the islands of Nantucket and Martha's Vineyard, including decisions at project inception about which types of disease resistance to pursue. This engagement process has prompted the researchers to use only white-footed mouse DNA if possible, meaning the current project will not involve gene drive. Instead, engineered mice would be released in the spring when the natural population is low, a plan unlikely to increase total numbers above the normal maximum in autumn. Community members are continually asked to share their suggestions and concerns, a process that has already identified potential ecological consequences unanticipated by the research team that will likely affect implementation. As an early example of CRISPR-based ecological engineering, Mice Against Ticks aims to start small and simple by working with island communities whose mouse populations can be lastingly immunized without gene drive. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.
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Casδ, an evolutionary transitional CRISPR system enables efficient genome editing across animals and plants.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) adaptive immune systems provide sequence-specific mechanisms for targeting foreign DNA or RNA and have been widely used in genome editing and DNA detection. Type V CRISPR-Cas systems are characterized by a single RNA-guided RuvC domain-containing effector, Cas12. Here, through comprehensive mining of large-scale genomic and metagenomic data from microbial sources, we identified a new Class 2 CRISPR-Cas effector superfamily, designated Casδ, comprising three members with protein sizes ranging from 867 to 936 amino acids. Biochemical analyses revealed that Casδ-1 functions as a single RNA-guided endonuclease with specific recognition of 5'-RYR-3' protospacer-adjacent motifs, where R represents A or G, and Y represents T or C. Casδ-1 exhibits robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity. Casδ-1 mediates efficient genome editing across species, achieving up to 60% indel rates in human cells while generating homozygous knockout lines in two agriculturally important monocot species (Oryza sativa and Zea mays) through stable transformation. Structural and evolutionary analyses reveal Casδ as an evolutionary transitional nuclease bridging Cas12n and canonical type V systems, featuring a C-terminal loop that is essential for activity. Collectively, Casδ is an evolutionarily distinct, compact (<1000 aa), tracrRNA-free CRISPR system enabling versatile cross-kingdom genome editing.
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Progress and challenges in applying CRISPR/Cas techniques to the genome editing of trees.
With the advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system, plant genome editing has entered a new era of robust and precise editing for any genes of interest. The development of various CRISPR/Cas toolkits has enabled new genome editing outcomes that not only target indel mutations but also enable base editing and prime editing. The application of the CRISPR/Cas toolkits has rapidly advanced breeding and crop improvement of economically important species. CRISPR/Cas toolkits have also been applied to a wide variety of tree species, including apple, bamboo, Cannabaceae, cassava, citrus, cacao tree, coffee tree, grapevine, kiwifruit, pear, pomegranate, poplar, ratanjoyt, and rubber tree. The application of editing to these species has resulted in significant discoveries related to critical genes associated with growth, secondary metabolism, and stress and disease resistance. However, most studies on tree species have involved only preliminary optimization of editing techniques, and a more in-depth study of editing techniques for CRISPR/Cas-based editing of tree species has the potential to rapidly accelerate tree breeding and trait improvements. Moreover, tree genome editing still relies mostly on Cas9-based indel mutation and Agrobacterium-mediated stable transformation. Transient transformation for transgene-free genome editing is preferred, but it typically has very low efficiency in tree species, substantially limiting its potential utility. In this work, we summarize the current status of tree genome editing practices using the CRISPR/Cas system and discuss limitations that impede the efficient application of CRISPR/Cas toolkits for tree genome editing, as well as future prospects.
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CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.
Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family.IMPORTANCEPlasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.
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Synthetic biology approaches for chromosomal integration of genes and pathways in industrial microbial systems.
Industrial biotechnology is reliant on native pathway engineering or foreign pathway introduction for efficient biosynthesis of target products. Chromosomal integration, with intrinsic genetic stability, is an indispensable step for reliable expression of homologous or heterologous genes and pathways in large-scale and long-term fermentation. With advances in synthetic biology and CRISPR-based genome editing approaches, a wide variety of novel enabling technologies have been developed for single-step, markerless, multi-locus genomic integration of large biochemical pathways, which significantly facilitate microbial overproduction of chemicals, pharmaceuticals and other value-added biomolecules. Notably, the newly discovered homology-mediated end joining strategy could be widely applicable for high-efficiency genomic integration in a number of homologous recombination-deficient microbes. In this review, we explore the fundamental principles and characteristics of genomic integration, and highlight the development and applications of targeted integration approaches in the three representative industrial microbial systems, including Escherichia coli, actinomycetes and yeasts.
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Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.
Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.
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VEGFA-Targeted M3-F4 Ionizable Lipid Nanoparticles Improve Diabetic Retinopathy.
Diabetic retinopathy (DR) is one of the leading causes of visual impairment and blindness worldwide. Current therapies for DR primarily focus on inhibiting vascular endothelial growth factor A (VEGFA); however, their efficacy remains limited due to drug resistance and the requirement for repeated intravitreal injections. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technology enables specific targeting and knockout of the VEGFA gene, offering a novel therapeutic approach for DR. In this study, we synthesized a novel ionizable lipid, M3, and assembled the optimal-performing M3-F4 into lipid nanoparticles (M3-F4 LNP) for codelivery of VEGFA-targeting Cas9 mRNA (mCas9) and single guide RNA (sgRNA). The optimized formulation, composed of M3:cholesterol:DSPC:DMG-PEG at a molar ratio of 45:42.5:10:2.5, exhibited a particle size below 100 nm, a PDI below 0.2, and an encapsulation efficiency above 80%. Sanger sequencing-based indel analysis confirmed VEGFA editing in HRMECs, with sgRNA1 achieving an indel frequency of approximately 28.7%. In high glucose-induced human retinal microvascular endothelial cells (HRMECs), the mCas9/sgVEGFA@M3-F4 LNP reduced cell proliferation, migration, invasion, and tube formation, while restoring endothelial barrier integrity and exerting anti-inflammatory effects. A single intravitreal injection of mCas9/sgVEGFA@M3-F4 LNP effectively inhibited pathological neovascularization and retinal leakage in both oxygen-induced retinopathy mice and streptozotocin-induced diabetic mice in vivo. Furthermore, it markedly attenuated VEGFA-induced inflammation while maintaining excellent biocompatibility. This study demonstrates M3-F4 LNP as a promising method for efficient CRISPR/Cas9 delivery and provides robust support for gene therapy strategies in DR treatment.
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Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.
SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.
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Genome editing strategies: potential tools for eradicating HIV-1/AIDS.
Current therapy for controlling human immunodeficiency virus (HIV-1) infection and preventing acquired immunodeficiency syndrome (AIDS) progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells, which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or "sterile" cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS.
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