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Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.
Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.
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Making sense of carbonic anhydrase function in zebrafish using antisense morpholinos.
Understanding gene function in vertebrate development requires tools that allow precise and timely manipulation of gene expression. Zebrafish (Danio rerio), with its transparent embryos and rapid development, offers an ideal model to study vertebrate biology. This review explores how morpholino oligonucleotides (MOs), a widely used tool for transient gene knockdown, have been employed to investigate the roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs) in zebrafish. CAs are metalloenzymes, while CARPs are inactive isozymes that play critical roles in pH regulation, ion transport, CO₂ metabolism, and protein interactions influencing diverse biological functions. Many of the MO knockdown studies presented here have been extensively conducted in our laboratory over the past decade, revealing novel roles for CAs in neural development, reproduction, and swim bladder formation. These studies also confirm roles previously reported in humans, such as pigmentation, acid-base homeostasis, neural development, and motor coordination. We discuss technical aspects of MO design, delivery, and validation, and address common challenges such as off-target effects, transient gene silencing, and the necessity of rescue experiments. In addition, the review includes a comparative analysis of MOs versus CRISPR/Cas9-based genome editing, underscoring their respective advantages and limitations for functional genomics. In conclusion, this review provides not only a methodological guide but also biological insights into CA function in zebrafish, highlighting how antisense technology continues to inform vertebrate development and disease modeling. The lessons learned here may inform the study of other gene families and support translational research in carbonic anhydrase-related human disorders.
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Optimization of lipid-assisted nanoparticle for disturbing neutrophils-related inflammation.
Inflammation is closely related to the development of many diseases and is commonly characterized by abnormal infiltration of immune cells, especially neutrophils. The current therapeutics of inflammatory diseases give little attention to direct modulation of these diseases with respect to immune cells. Nanoparticles are applied for efficient drug delivery into the disease-related immune cells, but their performance is significantly affected by their surface properties. In this study, to optimize the properties of nanoparticles for modulating neutrophils-related inflammation, we prepared a library of poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLANs) with different surface PEG density and surface charge. Optimized CLANs for neutrophils targeting were screened in high-fat diet (HFD)-induced type 2 diabetes (T2D) mice. Then, a CRISPR-Cas9 plasmid expressing a guide RNA (gRNA) targeting neutrophil elastase (NE) was encapsulated into the optimized CLAN and denoted as CLANpCas9/gNE. After intravenous injection, CLANpCas9/gNE successfully disrupted the NE gene of neutrophils and mitigated the insulin resistance of T2D mice via reducing the inflammation in epididymal white adipose tissue (eWAT) and in the liver. This strategy provides an example of abating the inflammatory microenvironment by directly modulating immune cells with nanoparticles carrying genome editing tools.
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Base editor enables rational genome-scale functional screening for enhanced industrial phenotypes in Corynebacterium glutamicum.
Genome-scale functional screening accelerates comprehensive assessment of gene function in cells. Here, we have established a genome-scale loss-of-function screening strategy that combined a cytosine base editor with approximately 12,000 parallel sgRNAs targeting 98.1% of total genes in Corynebacterium glutamicum ATCC 13032. Unlike previous data processing methods developed in yeast or mammalian cells, we developed a new data processing procedure to locate candidate genes by statistical sgRNA enrichment analysis. Known and novel functional genes related to 5-fluorouracil resistance, 5-fluoroorotate resistance, oxidative stress tolerance, or furfural tolerance have been identified. In particular, purU and serA were proven to be related to the furfural tolerance in C. glutamicum. A cloud platform named FSsgRNA-Analyzer was provided to accelerate sequencing data processing for CRISPR-based functional screening. Our method would be broadly useful to functional genomics study and strain engineering in other microorganisms.
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Advancements in CRISPR-based therapies for ocular pathologies: from disease mechanisms to intervention strategies.
Eye diseases caused by genetic mutations affect over 2.2 billion people worldwide. The development of CRISPR technology has opened exciting possibilities for how we diagnose and treat these conditions. However, designing effective CRISPR systems, managing potential risks, and considering the ethical questions around gene therapy in clinical practice are major challenges. To move forward successfully, it's important to evaluate how practical CRISPR-based treatments are for eye diseases from a clinical perspective, while also understanding how CRISPR systems work. In this review, we start by covering the basic principles behind CRISPR technology and explore its different types. Next, we look at various ways CRISPR is being used in eye research and treatments, from early studies to new clinical approaches. Lastly, we address the regulatory environment and ethical issues involved, discussing existing rules, safety concerns, and guidelines for genetic modifications in medical settings. Our goal is to share new insights into innovative treatments for eye diseases and to support the safe use of CRISPR in clinical eye care. This review aims to be a helpful resource for researchers, doctors, and regulators working on CRISPR-based therapies.
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Modification of the splice acceptor in CD163 exon 7 of pigs is insufficient to confer resistance to PRRSV.
CD163 is the primary receptor for PRRSV, and its SRCR5 domain, encoded by exon 7, is crucial for supporting PRRSV infection. Previous studies have used CRISPR/Cas9 technology to remove exon 7 from the host genome, and the edited pigs were completely resistant to PRRSV infection. In this study, we used CRISPR/Cas9 technology mimicking an adenine base editor (ABE) to edit the splice acceptor site of exon 7, rendering it nonfunctional. This alteration was intended to cause exon 6 to join directly to exon 8 during mRNA processing, resulting in a mature mRNA transcript that lacks exon 7, which encodes the SRCR5 domain. Piglets carrying the exon 7 splice site modification (CD163Ex7-ABE) were successfully generated. However, these pigs remained fully susceptible to infection with a PRRSV-2 isolate. Analysis of CD163 mRNA from the CD163Ex7-ABE pigs revealed that they predominantly expressed a mature CD163 mRNA lacking exon 7. However, due to cryptic splice sites, two additional mRNA isoforms were expressed, including an in-frame variant containing all of exon 7 and an extra 48 base pairs. This likely resulted in the expression of a full-length CD163 with a 16-amino-acid insertion upstream of the SRCR5 domain, which was sufficient to render the animals susceptible to PRRSV. Overall, our results demonstrate that merely modifying the splice acceptor site of CD163 exon 7 is not sufficient to generate PRRSV-resistant pigs.
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CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment.
The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not fully understood. In this study, based on a statistical mechanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong correlation between Cas9 cleavage efficiency and the stability of the DNA-RNA (R-loop) complex structures, with a Pearson correlation coefficient ranging from 0.775 to 0.886 for the tested systems. The finding leads to quantitative insights into important experimental results, such as the greater Cas9 tolerance to single-base mismatches in PAM-distal region than to PAM-proximal region and the high specificity and efficiency for shorter protospacers. Moreover, the results from the genome-wide off-target assessments, compared with other off-target scoring tools, indicate that the statistical mechanics-based approach provides more reliable off-target analyses and sgRNA design. To facilitate the genome engineering applications, a new web-based tool for genome-wide off-target assessment is established.
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Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA.
Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.
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Identifying Signalling Pathways Regulated by GPRC5B in β-Cells by CRISPR-Cas9-Mediated Genome Editing.
BACKGROUND/AIMS: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 β-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. METHODS: The optimal transfection reagent for use in MIN6 β-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 β-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 β-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. RESULTS: JetPRIME® was the best candidate for MIN6 β-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 β-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-β and IFNγ. CONCLUSION: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 β-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to β-cell proliferation and apoptosis.
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The transformational impact of site-specific DNA modifiers on biomedicine and agriculture.
The development of genetically modified livestock has been dependent on incremental technological advances such as embryo transfer, homologous recombination, and somatic cell nuclear transfer (SCNT). This development rate has increased exponentially with the advent of targeted gene modifiers such as zinc finger nucleases, TAL-effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas). CRISPR-Cas based systems in particular have broad applicability, and have low technical and economic barriers for their implementation. As a result, they are having, and will continue to have, a transformational impact in the field of gene editing in domestic animals. With these advances also comes the responsibility to properly apply this technology so it has a beneficial effect throughout all levels of society.
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