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Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck Tembusu virus infection.
Duck enteritis virus (DEV), duck tembusu virus (DTMUV), and highly pathogenic avian influenza virus (HPAIV) H5N1 are the most important viral pathogens in ducks, as they cause significant economic losses in the duck industry. Development of a novel vaccine simultaneously effective against these three viruses is the most economical method for reducing losses. In the present study, by utilizing a clustered regularly interspaced short palindromic repeats (CRISPR)/associated 9 (Cas9)-mediated gene editing strategy, we efficiently generated DEV recombinants (C-KCE-HA/PrM-E) that simultaneously encode the hemagglutinin (HA) gene of HPAIV H5N1 and pre-membrane proteins (PrM), as well as the envelope glycoprotein (E) gene of DTMUV, and its potential as a trivalent vaccine was also evaluated. Ducks immunized with C-KCE-HA/PrM-E enhanced both humoral and cell-mediated immune responses to H5N1 and DTMUV. Importantly, a single-dose of C-KCE-HA/PrM-E conferred solid protection against virulent H5N1, DTMUV, and DEV challenges. In conclusion, these results demonstrated for the first time that the CRISPR/Cas9 system can be applied for modification of the DEV genome rapidly and efficiently, and that recombinant C-KCE-HA/PrM-E can serve as a potential candidate trivalent vaccine to prevent H5N1, DTMUV, and DEV infections in ducks.
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DNA Base Editing Could Reverse Most Disease-Causing Point Mutations.
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Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology.
CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.
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Towards better base editing.
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Gene Therapy with CRISPR/Cas9 Coming to Age for HIV Cure.
The huge success of current antiretroviral therapy is mediated by a triple effect: (i) Halting progression to AIDS in infected persons; (ii) reducing the risk of transmission to contacts (treatment as prevention); and (iii) minimizing the risk of HIV acquisition treating uninfected persons at risk (pre-exposure prophylaxis). However, UNAIDS has estimated that only 70% of infected people globally are diagnosed, only 53% are treated, and overall 44% have undetectable viral load, which is the necessary request for ensuring any antiretroviral benefit. Thus, with 37 million people currently living with HIV worldwide and more than 2 million new infections per year, the prospects for global HIV eradication are far on the horizon. Over the past couple of years, rapid development has been seen for technologies enabling modification of gene expression, either by direct inhibition by RNA interference (RNAi) or by genomic modification at DNA level. In particular, genome-editing endonucleases have significantly improved our ability to make precise changes in the DNA of eukaryotic cells. Notably, firstgeneration genome-editing technologies (i.e., ZFNs and TALENs) have been replaced by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), which work with a short guide RNA (gRNA) to hybridize to a target DNA site and recruit the Cas9 endonuclease. Once integrated into the host genome, HIV gene expression is regulated by the LTR promoter. Hypothetically, gene editing of the HIV promoter might have the potential to deactivate viral transcription by the introduction of mutations or fragment excision. HIV gene therapy progressed very slowly until recent breakthroughs in gene-editing methods using CRISPR/Cas9 (Liao et al. Nat Commun 2015;6:6413). Using a shorter version of the Cas9 endonuclease ensembled into an adenoviral vector, critical segments of thAQ!e viral DNA genome spanning between the LTR and gag regions were successfully removed in HIV transgenic mice. Excision was confirmed in all examined tissues as well as in circulating lymphocytes and resulted in a drastic reduction of HIV-RNA (Kaminski et al. Gene Ther 2016;23:690-5). Moreover, using latently infected CD4+ T lymphocytes from HIV-infected persons, lentiviral-delivered CRISPR/Cas9 precisely removed the entire HIV genome spanning between the 50 and 30 LTRs of integrated HIV proviral DNA (Kaminski et al., Sci Rep 2016;6:22555), providing a proof of concept of the high potential of genome-editing technologies. Before moving to the clinic, the CRISPR/Cas9 technology must solve several major issues in the HIV scenario. First, generation of resistance is a major concern. Mutations may occur surrounding the targeted site and result in the selection of strains that are no longer recognized nor cleaved by CRISPR (Badia et al. Curr Opin Virol 2017;24:46-54). The efficacy of the anti-HIV CRISPR/Cas9 strategy is highly dependent on the gRNA sequence, yet some mutant viral strains show poor or no cleavage at all. Higher CRISPR/Cas9 pressure could delay but not eliminate viral replication when using a combination of distinct gRNAs targeting distinct HIV proviral genes. In this case, although the reading frame may remain unaltered, an accumulation of insertions and/or deletions may occur in the target sequence, rendering new viral strains insensitive to CRISPR/Cas9 cleavage. Finally, double-strand breaks resulting from CRISPR/Cas9 activity and subsequent cellular non-homologous end joining machinery may introduce mutations in sequences that are no longer recognized by the gRNA, and therefore not susceptible to Cas9 cleavage. A second consideration is a need for developing safe and effective mechanisms of delivery. Adenoviral vectors have long been studied in gene therapy and represent an ideal viral vector for transduction at different tissues. However, the packaging size of adenoviral vectors is a limiting factor, especially for CRIPSR/Cas9. Third, HIV has a genome of about 10 kb while a gRNA generally only targets 20 bp of the DNA molecule, which means that there are thousands available targeting sites for the provirus in latently infected cells. To date, there is no platform established solely for gRNA candidate evaluation in HIV provirus eradication. A final consideration is an access to all tissues and cells potentially harboring the HIV provirus, including reservoirs as the central nervous system. In this regard, efforts are being focused in the development of Cas9/gRNA nanoparticle formulations. To overcome these problems, a group in Florida recently developed human transgenic cells that may be used for gene-editing studies and as platform for high-throughput screen of HIV provirus disrupters (Huang et al. Sci Rep 2017;7:5955). Of note, Cas9 protein instead of a Cas9 plasmid was used. Compared to a plasmid introduction, Cas9 protein agents could be easily quantitatively applied and standardized, mimicking better real clinic scenarios. In summary, RNAi-based technologies have widely dominated gene therapy research during the past decade, with overall slow progress. However, the advent of new gene-editing technologies, and especially the CRISPR/Cas9 system, has revolutionized the field. In the HIV context, CRISPR/Cas9 applications might go further than those of RNAi, for example, enabling excision of segments of integrated proviral DNA from latently infected cells and allowing complete provirus elimination, or it may be used to reverse HIV latency. Although important challenges still need to be overcome, a promising pathway to HIV cure seems to have been found.
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A novel mouse model of cerebral microbleeds by targeted Col4a1 editing in adult brain microvessels.
Cerebral small vessel disease is a leading cause of cognitive decline and stroke in the elderly, with cerebral microbleeds (CMBs) serving as a key imaging biomarker. Despite their clinical significance, the pathophysiological mechanisms underlying cerebral small vessel disease remain poorly understood owing to a lack of appropriate animal models. We performed targeted deletion of Col4a1 in brain microvessels of adult mice using brain endothelium-specific adeno-associated virus (AAV)-BR1 vectors with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). Eight-week-old Cas9 transgenic mice received retro-orbital injections of AAV-BR1 containing single guide RNA (sgRNA) targeting Col4a1 or control Rosa26 sequences. Animals underwent longitudinal behavioural testing, including novel object recognition, Y-maze and rotarod tests, over 6 months. Brain pathology was assessed using T2*-weighted MRI, histological analysis and electron microscopy. For human studies, we analysed MRI and genomic data from 836 participants from the BICWALZS biobank, examining associations between genetic variants and CMB burden using linear regression and χ2 analyses. T2*-weighted MRI revealed numerous CMBs with distributions remarkably similar to human CMBs, appearing within 3 months post-injection. CMB burden increased progressively over 6 months in a dose-dependent manner. Behaviourally, mice exhibited progressive cognitive decline and motor incoordination. Histological examinations revealed haemosiderin deposits corresponding to MRI-detected CMBs, without macroscopic intracerebral haemorrhage or white matter changes. Ultrastructural analysis demonstrated significant basement membrane thinning in Col4a1-depleted microvessels. CMB accumulation was associated with widespread astrocytic reactivity extending beyond microbleed sites, whereas microglial activation remained localized. In human subjects, we identified significant associations between four genetic variants of TIMP2, an endogenous inhibitor of the matrix-degrading enzyme MMP2 and CMB burden, with odds ratios of 1.50-1.96 for increased microbleed susceptibility. This work provides the first animal model demonstrating that selective disruption of collagen IV in adult brain microvessels is sufficient to generate CMBs with high penetrance and dose-dependent tunability. Our findings establish that basement membrane integrity is critical for preventing microbleed formation and suggest that dysregulated collagen IV homeostasis underlies sporadic human CMB development.
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Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors.
The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34⁺ HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.
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BES-Designer: A Web Tool to Design Guide RNAs for Base Editing to Simplify Library.
CRISPR/Cas base editors offer precise conversion of single nucleotides without inducing double-strand breaks. This technology finds extensive applications in gene therapy, gene function analysis, and other domains. However, a crucial challenge lies in selecting the appropriate guide RNAs (gRNAs) for base editing. Although various gRNAs design tools exist, creating a simplified base-editing library with diverse protospacer adjacent motifs (PAM) sequences for gRNAs screening remains a challenge. We present a user-friendly web tool, BES-Designer ( https://bes-designer.aielab.net ), for gRNAs design based on base editors, aimed at streamlining the creation of a base-editing library. BES-Designer incorporates our proposed rules for target sequence simplification, helping researchers narrow down the scope of biological experiments in the lab. It allows users to design target sequences with various PAMs and editing types simultaneously, and prioritize them in the simplified base-editing library. This tool has been experimentally proven to achieve a 30% simplification efficiency on the base-editing-library.
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Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion.
Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.
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Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.
CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.
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