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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 12 Nov 2024 at 01:32 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2024-11-06

Lou H, Xiang H, Zeng W, et al (2024)

Protocol for transformation-free genome editing in plants using RNA virus vectors for CRISPR-Cas delivery.

STAR protocols, 5(4):103437 pii:S2666-1667(24)00602-6 [Epub ahead of print].

Plant virus vectors have emerged as promising tools for CRISPR-Cas reagent delivery. Here, we present a protocol for DNA-free plant genome editing using an engineered RNA virus vector for the transient delivery of CRISPR-Cas components. We describe steps for viral vector construction, viral vector recovery through agroinoculation of Nicotiana benthamiana, mechanical inoculation of target plant hosts, analysis of somatic mutagenesis frequency, and regeneration of mutant plants. The method achieves high editing efficiency and eliminates the need for stable plant transformation. For complete details on the use and execution of this protocol, please refer to Liu et al.[1].

RevDate: 2024-11-06

Xiao W, Weissman JL, PLF Johnson (2024)

Ecological drivers of CRISPR immune systems.

mSystems [Epub ahead of print].

UNLABELLED: CRISPR-Cas is the only known adaptive immune system of prokaryotes. It is a powerful defense system against mobile genetic elements such as bacteriophages. While CRISPR-Cas systems can be found throughout the prokaryotic tree of life, they are distributed unevenly across taxa and environments. Since adaptive immunity is more useful in environments where pathogens persist or reoccur, the density and/or diversity of the host/pathogen community may drive the uneven distribution of CRISPR systems. We directly tested hypotheses connecting CRISPR incidence with prokaryotic density/diversity by analyzing 16S rRNA and metagenomic data from publicly available environmental sequencing projects. In terms of density, we found that CRISPR systems are significantly favored in lower abundance (less dense) taxa and disfavored in higher abundance taxa, at least in marine environments. When we extended this work to compare taxonomic diversity between samples, we found CRISPR system incidence strongly correlated with diversity in human oral environments. Together, these observations confirm that, at least in certain types of environments, the prokaryotic ecological context indeed plays a key role in selecting for CRISPR immunity.

IMPORTANCE: Microbes must constantly defend themselves against viral pathogens, and a large proportion of prokaryotes do so using the highly effective CRISPR-Cas adaptive immune system. However, many prokaryotes do not. We investigated the ecological factors behind this uneven distribution of CRISPR-Cas immune systems in natural microbial populations. We found strong patterns linking CRISPR-Cas systems to prokaryotic density within ocean environments and to prokaryotic diversity within human oral environments. Our study validates previous within-lab experimental results that suggested these factors might be important and confirms that local environment and ecological context interact to select for CRISPR immunity.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Cao Y, Lin H, Lu X, et al (2025)

Benchtop to at-home test: Amplicon-depleted CRISPR-regulated loop mediated amplification at skin-temperature for viral load monitoring.

Biosensors & bioelectronics, 267:116866.

CoRPLA (CRISPR-regulated One-pot Recombinase Polymerase Loop-mediated Amplification) is an amplicon-depleted skin-temperature operated iNAAT designed for at-home testing. It uses specially designed loop primers to enhance isothermal amplification, triggering Cas12 for in-situ amplicon depletion and signal amplification. This method addresses issues like amplicon-derived aerosol contamination and complex assay formats, enabling quantitative detection with sub-attomolar sensitivity (0.5 cps/μL). CoRPLA employs a DNA hydrogel wearable tape for real-time, colorimetric readout, allowing visual differentiation of pathogen loads. It was validated with clinical samples for SARS-CoV-2, RSV, influenza A, and HPV, successfully identifying multi-level viral loads of the positive cases with results consistent with qPCR. Offering high sensitivity while eliminating false positives from aerosol contamination, CoRPLA bridges the molecular assay from benchtop to home for daily viral infections monitoring.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Li L, Feng C, Zhang W, et al (2024)

Mitigation of Cisplatin-Induced Nephrotoxicity and Augmentation of Anticancer Potency via Tea Polyphenol Nanoparticles' Codelivery of siRNA from CRISPR/Cas9 Screened Targets.

ACS applied materials & interfaces, 16(44):59721-59737.

Cisplatin, a frontline chemotherapeutic agent against cancer, faces challenges in clinical application due to significant toxicities and suboptimal efficacy. Renal toxicity, a dose-limiting factor of cisplatin, results from multifactorial processes including cisplatin-induced cellular pyroptosis, oxidative damage, and inflammatory responses. Our findings reveal that Tea Polyphenols Nanoparticles (TPNs) derived from Epigallocatechin gallate (EGCG) effectively could address these diverse mechanisms, comprehensively alleviating cisplatin-induced nephrotoxicity. Leveraging TPNs as carriers, chemical conjugation enables the encapsulation of tetravalent cisplatin prodrug, extending its systemic half-life, enhancing tumor tissue accumulation, while simultaneously mitigating renal toxicity. Concurrently, employing a CRISPR/Cas9 kinase library, we identified CSNK2A1 as a target sensitizing tumor cells to cisplatin, enabling specific siRNA sequences to augment cisplatin susceptibility, thereby minimizing the dosage requirement. Benefiting from the versatile carrier properties of TPNs to codeliver cisplatin prodrug and anti-CSNK2A1 siRNA, we developed a codelivery system, Pt-TPNs/siRNA. Pt-TPNs/siRNA not only enhances the anticancer effects but also mitigates cisplatin-induced renal toxicity, achieving efficacy while reducing toxicity. Mechanistic and safety assessments of these nanoparticles were conducted at both cellular and animal levels, opening new avenues for improved clinical utilization of cisplatin.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Hwang I, Song YH, S Lee (2025)

Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products.

Biosensors & bioelectronics, 267:116859.

The CRISPR-Cas12a system has emerged as a promising tool for molecular diagnostics due to its indiscriminate trans-ssDNase activity. However, the sensitivity of Cas12a-based diagnostics remains insufficient for clinical use without a pre-amplification step such as loop-mediated isothermal amplification, and therefore the trans-cleavage activity of Cas12a needs to be enhanced. Here, we present a novel strategy to enhance the trans-cleavage activity of Cas12a by reducing the steric hindrance from cis-cleavage products. We have designed Cas12a variants with alanine mutations in the target strand loading (TSL) domain, resulting in reduced affinity for target strand (TS) overhangs to the catalytic site and significantly increased trans-cleavage efficiency by up to 5.8-fold. In addition, we used a novel salt dilution method to exploit the enhanced trans-cleavage activity of Cas12a under low ionic strength conditions (7-fold), significantly improving the sensitivity of our Cas12a-based detection system. To demonstrate the clinical potential of our Cas12a-based detection system, we validated its ability to detect small amounts of hepatitis B virus (HBV) DNA model using the combination of the KE1096AA Cas12a mutant and the salt dilution method, which enables the detection of DNA at atto-molar concentrations. Our strategy to enhance the trans-cleavage activity of Cas12a paves the way for the development of more sensitive and efficient Cas12a-based diagnostics.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Hu Y, Liao Y, Pan S, et al (2025)

A Triple-Mismatch Differentiating assay exploiting activation and trans cleavage of CRISPR-Cas12a for mutation detection with ultra specificity and sensitivity.

Biosensors & bioelectronics, 267:116826.

Liquid biopsy technology is non-invasive and convenient, and is currently an emerging technology for cancer screening. Among them, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 12a (Cas12a) based nucleic acid detection technology has the advantages of high sensitivity, rapidity, and easy operation. However, CRISPR-Cas12a does not discriminate single-base mismatches of targets well enough to meet the needs of clinical detection. Herein, we developed the Triple-Mismatch Differentiating (TMD) assay. This assay amplified the small thermodynamic difference in mismatches at one site at the level of CRISPR-Cas12a activation to a significant thermodynamic difference at three sites at both the level of CRISPR-Cas12a activation and trans-cleavage, which greatly improves the ability of CRISPR-Cas12a to discriminate between base mismatches. Our manipulation greatly improved the specificity of the CRISPR-Cas12a system while maintaining its inherent sensitivity and simplicity, increasing the detection limit to 0.0001%. When testing samples from pancreatic cancer patients, our results were highly consistent with NGS sequencing results. We believe that the TMD assay will provide a new technology for early cancer detection and will be widely used in the clinical practice.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Wang K, Yin H, Li S, et al (2025)

Quantitative detection of circular RNA and microRNA at point-of-care using droplet digital CRISPR/Cas13a platform.

Biosensors & bioelectronics, 267:116825.

Circular RNA (circRNA) and microRNA (miRNA) are both non-coding RNAs (ncRNAs) that serve as biomarkers for cancer diagnosis and prognosis. Quantitative detection of these ncRNAs is of particular importance to elucidate the functional mechanisms and evaluate their potential as biomarkers. However, the inherent structures of circRNA and miRNA are different from the mRNA, conventional qRT-PCR is unsuitable for the detection of these ncRNAs. Here, we propose a sensitive method for quantitative detection of circRNA and miRNA using polydisperse droplet digital CRISPR/Cas13a (PddCas13a). It can achieve limits of detection (LOD) as low as ∼10 aM without polymerase-based amplification. To efficiently detect the circRNA and miRNA in real samples, we use a chemically modified crRNA to enhance the stability of crRNA and improve the performance of Cas13a in complex environments containing contaminants. By integrating an extraction-free procedure with PddCas13a, we experimentally demonstrate the applicability of PddCas13a by testing clinical samples. Furthermore, we develop an automated and portable instrument for PddCas13a and verify its applicability for the detection of circRNA and miRNA from exosomes in point-of-care (POC) setting. This is the first report to detect the circRNA and miRNA simultaneously in POC setting. We envision this platform could promote the research of ncRNAs.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Marquart KF, Mathis N, Mollaysa A, et al (2024)

Effective genome editing with an enhanced ISDra2 TnpB system and deep learning-predicted ωRNAs.

Nature methods, 21(11):2084-2093.

Transposon (IS200/IS605)-encoded TnpB proteins are predecessors of class 2 type V CRISPR effectors and have emerged as one of the most compact genome editors identified thus far. Here, we optimized the design of Deinococcus radiodurans (ISDra2) TnpB for application in mammalian cells (TnpBmax), leading to an average 4.4-fold improvement in editing. In addition, we developed variants mutated at position K76 that recognize alternative target-adjacent motifs (TAMs), expanding the targeting range of ISDra2 TnpB. We further generated an extensive dataset on TnpBmax editing efficiencies at 10,211 target sites. This enabled us to delineate rules for on-target and off-target editing and to devise a deep learning model, termed TnpB editing efficiency predictor (TEEP; https://www.tnpb.app), capable of predicting ISDra2 TnpB guiding RNA (ωRNA) activity with high performance (r > 0.8). Employing TEEP, we achieved editing efficiencies up to 75.3% in the murine liver and 65.9% in the murine brain after adeno-associated virus (AAV) vector delivery of TnpBmax. Overall, the set of tools presented in this study facilitates the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics.

RevDate: 2024-11-07
CmpDate: 2024-11-07

Fu J, Mo R, Li Z, et al (2025)

An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR/Cas12a.

Biosensors & bioelectronics, 267:116740.

Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.

RevDate: 2024-11-06

Cheng H, Deng H, Ma D, et al (2024)

Insight into the natural regulatory mechanisms and clinical applications of the CRISPR-Cas system.

Heliyon, 10(20):e39538.

CRISPR-Cas, the adaptive immune system exclusive to prokaryotes, confers resistance against foreign mobile genetic elements. The CRISPR-Cas system is now being exploited by scientists in a diverse range of genome editing applications. CRISPR-Cas systems can be categorized into six different types based on their composition and mechanism, and there are also natural regulatory biomolecules in bacteria and bacteriophages that can either enhance or inhibit the immune function of CRISPR-Cas. The CRISPR-Cas systems are currently being trialed as a new tool for gene therapy to treat various human diseases, including cancers and genetic diseases, offering significant therapeutic potential. This paper comprehensively summarizes various aspects of the CRISPR-Cas system, encompassing its diversity, regulatory mechanisms, its clinical applications and the obstacles encountered.

RevDate: 2024-11-05

Anonymous (2024)

Ancient and versatile CRISPR-Cas nuclease created with ancestral sequence reconstruction.

Nature biotechnology [Epub ahead of print].

RevDate: 2024-11-05
CmpDate: 2024-11-05

Endo M, Negishi K, S Toki (2025)

Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.

Methods in molecular biology (Clifton, N.J.), 2869:101-111.

Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM. Then, engineered and natural Cas variants with altered PAM recognition are used for base editor to expand the flexibility of base substitution position. In this chapter, we describe a protocol for base editing based on SpCas9-NG, which is a rationally engineered SpCas9 variant that can recognize relaxed NG PAMs.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Nishizawa-Yokoi A, S Toki (2025)

Gene Targeting via CRISPR/Cas9-Mediated DNA Double-Strand Break Induction in Rice.

Methods in molecular biology (Clifton, N.J.), 2869:91-100.

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination. However, due to its low frequency, it has proved difficult to establish a universal GT system. The availability of the CRISPR/Cas9 system for genome editing in plants has opened up possibilities to apply GT successfully to some plant species. Here, we provide protocols for CRISPR/Cas9-mediated DNA double-strand break (DSB)-induced GT in rice.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Sukegawa S, Toki S, H Saika (2025)

Agrobacterium-Mediated Transformation and Targeted Mutagenesis Using SpCas12f in Rice.

Methods in molecular biology (Clifton, N.J.), 2869:75-90.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) is an adaptive prokaryote immune system against foreign DNA/RNA that is now applied widely to genome editing. A miniature Cas, CRISPR-Cas12f, is one-half to one-third the size of the CRISPR-Cas9 that is commonly used in genome editing experiments in many organisms, including higher plants. The compactness of CRISPR-Cas12f is expected to be advantageous in terms of vector construction and transformation frequency. Moreover, CRISPR-Cas12f can be useful for virus vector-mediated genome editing because the size of the transgene is the major restriction in the use of virus vectors. Here, we describe our protocol for targeted mutagenesis using Cas12f derived from Syntrophomonas palmitatica (SpCas12f) via Agrobacterium-mediated transformation in rice. We also summarize some approaches to improve the frequency of targeted mutagenesis using SpCas12f.

RevDate: 2024-11-06
CmpDate: 2024-11-06

Qiao Y, Wang X, Fan Z, et al (2024)

Raman-enhanced sensor based on CRISPR-SERS technology for the rapid and hypersensitive detection of Mycobacterium tuberculosis.

Analytical and bioanalytical chemistry, 416(28):6551-6562.

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R[2] = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.

RevDate: 2024-11-06
CmpDate: 2024-11-06

Haga K, Tokui T, Miyamoto K, et al (2024)

Neonatal Fc receptor is a functional receptor for classical human astrovirus.

Genes to cells : devoted to molecular & cellular mechanisms, 29(11):983-1001.

Human astrovirus (HAstV) is a global cause of gastroenteritis in infants, the elderly, and the immunocompromised. However, the molecular mechanisms that control its susceptibility are not fully understood, as the functional receptor used by the virus has yet to be identified. Here, a genome-wide CRISPR-Cas9 library screen in Caco2 cells revealed that the neonatal Fc receptor (FcRn) can function as a receptor for classical HAstV (Mamastrovirus genotype 1). Deletion of FCGRT or B2M, which encode subunits of FcRn, rendered Caco2 cells and intestinal organoid cells resistant to HAstV infection. We also showed that human FcRn expression renders non-susceptible cells permissive to viral infection and that FcRn binds directly to the HAstV spike protein. Therefore, our findings provide insight into the entry mechanism of HAstV into susceptible cells. We anticipate that this information can be used to develop new therapies targeting human astroviruses, providing new strategies to treat this global health issue.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Punetha M, Saini S, Sharma S, et al (2024)

CRISPR-Mediated SRY Gene Mutation Increases the Expression of Female Lineage-Specific Gene in Pre-Implantation Buffalo Embryo.

Reproduction in domestic animals = Zuchthygiene, 59(11):e14739.

In mammals, sex determination is governed by the SRY gene on the Y chromosome, redirecting gonadal development from forming ovaries to testes. Mutations or alterations in the SRY gene can significantly affect phenotypic changes and lineage-specific markers. This study aims to elucidate the role of the SRY gene in buffalo embryos using CRISPR-Cas9 technology. We designed a crRNA targeting the HMG domain of the SRY gene using the CRISPOR algorithm. Nucleofection of sgRNA-Cas9 RNPs into buffalo fibroblasts confirmed efficient cleavage at the targeted site. Using this validated guide, we investigated the role of the SRY gene in sexual determination by electroporating CRISPR-Cas9-RNPs into single-stage zygotes of buffalo. Genetic changes in the SRY gene were confirmed through sequencing, revealing mosaic blastocysts with multiple alleles and non-mosaic mutants. Mutations in SRY gene increased the expression of female lineage-specific gene Wnt4 whereas decreased the expression of male specific gene SOX9 in blastocysts, suggesting reprogramming towards female sex determination pathways. Our findings provide insights into buffalo sex differentiation mechanisms and potential applications in reproductive strategies for breeding programmes.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Martinez R, Finocchiaro C, Delhaye L, et al (2024)

A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens.

Frontiers in immunology, 15:1444886.

Cancer cells effectively evade immune surveillance, not only through the well-known PD-1/PD-L1 pathway but also via alternative mechanisms that impair patient response to immune checkpoint inhibitors. We present a novel co-culture model that pairs a reporter T-cell line with different melanoma cell lines that have varying immune evasion characteristics. We developed a scalable high-throughput lentiviral arrayed CRISPR interference (CRISPRi) screening protocol to conduct gene perturbations in both T-cells and melanoma cells, enabling the identification of genes that modulate tumor immune evasion. Our study functionally validates the co-culture model system and demonstrates the performance of the CRISPRi-screening protocol by modulating the expression of known regulators of tumor immunity. Together, our work provides a robust framework for future research aimed at systematically exploring mechanisms of tumor immune evasion.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Borah A, Singh S, Chattopadhyay R, et al (2024)

Integration of CRISPR/Cas9 with multi-omics technologies to engineer secondary metabolite productions in medicinal plant: Challenges and Prospects.

Functional & integrative genomics, 24(6):207.

Plants acts as living chemical factories that may create a large variety of secondary metabolites, most of which are used in pharmaceutical products. The production of these secondary metabolites is often much lower. Moreover, the primary constraint after discovering potential metabolites is the capacity to manufacture sufficiently for use in industrial and therapeutic contexts. The development of omics technology has brought revolutionary discoveries in various scientific fields, including transcriptomics, metabolomics, and genome sequencing. The metabolic pathways leading to the utilization of new secondary metabolites in the pharmaceutical industry can be identified with the use of these technologies. Genome editing (GEd) is a versatile technology primarily used for site-directed DNA insertions, deletions, replacements, base editing, and activation/repression at the targeted locus. Utilizing GEd techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9), metabolic pathways engineered to synthesize bioactive metabolites optimally. This article will briefly discuss omics and CRISPR/Cas9-based methods to improve secondary metabolite production in medicinal plants.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Zhang Y, Liu Y, Qin W, et al (2024)

Cytosine base editors with increased PAM and deaminase motif flexibility for gene editing in zebrafish.

Nature communications, 15(1):9526.

Cytosine base editing is a powerful tool for making precise single nucleotide changes in cells and model organisms like zebrafish, which are valuable for studying human diseases. However, current base editors struggle to edit cytosines in certain DNA contexts, particularly those with GC and CC pairs, limiting their use in modelling disease-related mutations. Here we show the development of zevoCDA1, an optimized cytosine base editor for zebrafish that improves editing efficiency across various DNA contexts and reduces restrictions imposed by the protospacer adjacent motif. We also create zevoCDA1-198, a more precise editor with a narrower editing window of five nucleotides, minimizing off-target effects. Using these advanced tools, we successfully generate zebrafish models of diseases that were previously challenging to create due to sequence limitations. This work enhances the ability to introduce human pathogenic mutations in zebrafish, broadening the scope for genomic research with improved precision and efficiency.

RevDate: 2024-11-04

Trivedi V, Mohseni A, Lonardi S, et al (2024)

Balanced Training Sets Improve Deep Learning-Based Prediction of CRISPR sgRNA Activity.

ACS synthetic biology [Epub ahead of print].

CRISPR-Cas systems have transformed the field of synthetic biology by providing a versatile method for genome editing. The efficiency of CRISPR systems is largely dependent on the sequence of the constituent sgRNA, necessitating the development of computational methods for designing active sgRNAs. While deep learning-based models have shown promise in predicting sgRNA activity, the accuracy of prediction is primarily governed by the data set used in model training. Here, we trained a convolutional neural network (CNN) model and a large language model (LLM) on balanced and imbalanced data sets generated from CRISPR-Cas12a screening data for the yeast Yarrowia lipolytica and evaluated their ability to predict high- and low-activity sgRNAs. We further tested whether prediction performance can be improved by training on imbalanced data sets augmented with synthetic sgRNAs. Lastly, we demonstrated that adding synthetic sgRNAs to inherently imbalanced CRISPR-Cas9 data sets from Y. lipolytica and Komagataella phaffii leads to improved performance in predicting sgRNA activity, thus underscoring the importance of employing balanced training sets for accurate sgRNA activity prediction.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Ghane A, Malhotra PK, Sanghera GS, et al (2024)

CRISPR/Cas technology: fueling the future of Biofuel production with sugarcane.

Functional & integrative genomics, 24(6):205.

The objective of present review is to provide a scientific overview of sugarcane as a potential feedstock for biofuel and use of genome editing approach for improvement of industrial and agronomical traits in sugarcane. Sugarcane, a perennial tropical grass with a high biomass index, is a promising feedstock for bioethanol production, and its bagasse, rich in lignocellulosic material, serves as an ideal feedstock for producing second-generation bioethanol. To improve the conversion of sugarcane biomass into biofuels, developing varieties with improved biomass degradability and high biomass and sucrose content is essential. The complex genome architecture and earlier lack of sequence data hindered biotechnological advancements in sugarcane, but recent genome sequence updates offer new opportunities for sugarcane improvement. The first genetically modified sugarcane was developed in 1992 by Bower and Birch using microprojectile bombardment of embryogenic callus. Since then, transgenic techniques have rapidly evolved, leading to the advancement of genome editing technologies. Application of genome editing tools particularly CRISPR/Cas system has been successfully used in sugarcane for editing. Recently, multiple alleles of the magnesium chelatase and acetolactate synthase genes in sugarcane have been successfully edited through multiplexing. Additionally, CRISPR-edited sugarcane varieties with modified cell wall components and increased sucrose content for enhanced bioethanol production have been developed. At the end, the future of CRISPR-edited crops will depend on how well regulatory frameworks adapt to the rapidly evolving technology.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Son H, Kang Y, Song YH, et al (2024)

Effects of steric hindrance from single-stranded overhangs on target-strand loading into the Cas12a active site.

Chemical communications (Cambridge, England), 60(89):13087-13090.

CRISPR-Cas12a, an RNA-guided DNA endonuclease, induces double-strand breaks by cleaving the non-target strand (NTS) first, followed by the target strand (TS). Using single-molecule FRET with alternating-laser excitation, we found that steric hindrance from the 3' overhangs of both the cleaved NTS and crRNA impedes TS loading into the catalytic core. Our study highlights the direct involvement of both 3' NTS and crRNA overhangs in TS cleavage, offering insights into regulatory strategies for Cas12a cleavage reactions.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Wang X, Yang T, Zhang Y, et al (2024)

Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR/Cas12a and Recombinase Polymerase Amplification.

Analytical chemistry, 96(44):17567-17575.

Conventional methods for detecting single nucleotide polymorphisms (SNPs) in clinical practice often require substantial time, labor, and specialized equipment, limiting their widespread application. To address this limitation, we refined our previous SNP detection method, IMAS-RPA [introducing an extra mismatched base adjacent to the single-base mutant site by recombinase polymerase amplification (RPA)], resulting in an updated version termed IMAS-RPAv2. We began by introducing a suboptimal protospacer adjacent motif (PAM) sequence, GTTG, into the double-stranded DNA (dsDNA) products using either RPA or reverse transcription RPA. This modification decreased the efficiency with which CRISPR RNA (crRNA) recognizes the PAM and locally unwinds the dsDNA to form an R loop. After a delay, the R loop forms. However, due to the intentional incorporation of a mismatched base on the crRNA relative to the wild-type double-stranded DNA (WT-dsDNA), a continuous two-base mismatch is established between the crRNA and WT-dsDNA. Consequently, WT-dsDNA does not activate CRISPR/Cas12a's cleavage activity within a short time, while variant-type dsDNA continues to activate CRISPR/Cas12a and produce a robust fluorescence signal. This improvement significantly enhances the SNP discrimination sensitivity, allowing for detection at the single-copy level. Results were observed using both a conventional microplate reader and a specially designed portable device created through 3D printing. This device allows a direct fluorescence observation without the need for additional equipment. Consequently, the entire detection process becomes independent of large-scale equipment. This greatly expands its range of applications and offers promising prospects for clinical use.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Li L, Li M, Wang S, et al (2024)

Development of a CRISPR/Cas12a-facilitated fluorescent aptasensor for sensitive detection of small molecules.

International journal of biological macromolecules, 280(Pt 4):136041.

The integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) exhibits superior performance in biosensor construction. And the distinctive role of aptamers in target recognition has long been a focal point of research. Through the combination of Cas12a with cis-cleavage activity and aptamer with specific recognition, a simple and rapid fluorescent biosensor has been constructed. Interestingly, with modified fluorescent and quenching groups at two ends, aptamers play a dual role: primarily as the elements for target recognition and additionally functioning act as the fluorescent probe for signal output. Coupling with cis-cleavage of Cas12a, the demand of additional signal probes is eliminated, thus simplifying the reaction system and enhancing result accuracy. Taking okadaic acid (OA) as a representative small molecule model to evaluate the sensor's performance, a simple and straightforward detection method was established. Following this, the universality of the constructed fluorescent aptasensor was validated by incorporating an adenosine triphosphate (ATP) aptamer. Consequently, the CRISPR/Cas12a-assisted aptasensor was demonstrated to serve as a versatile detection platform for small molecules in food safety and clinical diagnostics. In the forthcoming research endeavors, it can be further extended for applications in environmental analysis and various other fields.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Yao B, Lei Z, Gonçalves MAFV, et al (2024)

Integrating Prime Editing and Cellular Reprogramming as Novel Strategies for Genetic Cardiac Disease Modeling and Treatment.

Current cardiology reports, 26(11):1197-1208.

PURPOSE OF REVIEW: This review aims to evaluate the potential of CRISPR-based gene editing tools, particularly prime editors (PE), in treating genetic cardiac diseases. It seeks to answer how these tools can overcome current therapeutic limitations and explore the synergy between PE and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for personalized medicine.

RECENT FINDINGS: Recent advancements in CRISPR technology, including CRISPR-Cas9, base editors, and PE, have demonstrated precise genome correction capabilities. Notably, PE has shown exceptional precision in correcting genetic mutations. Combining PE with iPSC-CMs has emerged as a robust platform for disease modeling and developing innovative treatments for genetic cardiac diseases. The review finds that PE, when combined with iPSC-CMs, holds significant promise for treating genetic cardiac diseases by addressing their root causes. This approach could revolutionize personalized medicine, offering more effective and precise treatments. Future research should focus on refining these technologies and their clinical applications.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Qi Z, Meng X, Xu M, et al (2024)

A novel Pik allele confers extended resistance to rice blast.

Plant, cell & environment, 47(12):4800-4814.

In the ongoing arms race between rice and Magnaporthe oryzae, the pathogen employs effectors to evade the immune response, while the host develops resistance genes to recognise these effectors and confer resistance. In this study, we identified a novel Pik allele, Pik-W25, from wild rice WR25 through bulked-segregant analysis, creating the Pik-W25 NIL (Near-isogenic Lines) named G9. Pik-W25 conferred resistance to isolates expressing AvrPik-C/D/E alleles. CRISPR-Cas9 editing was used to generate transgenic lines with a loss of function in Pik-W25-1 and Pik-W25-2, resulting in loss of resistance in G9 to isolates expressing the three alleles, confirming that Pik-W25-induced immunity required both Pik-W25-1 and Pik-W25-2. Yeast two-hybrid (Y2H) and split luciferase complementation assays showed interactions between Pik-W25-1 and the three alleles, while Pik-W25-2 could not interact with AvrPik-C, -D, and -E alleles with Y2H assay, indicating Pik-W25-1 acts as an adaptor and Pik-W25-2 transduces the signal to trigger resistance. The Pik-W25 NIL exhibited enhanced field resistance to leaf and panicle blast without significant changes in morphology or development compared to the parent variety CO39, suggesting its potential for resistance breeding. These findings advance our knowledge of rice blast resistance mechanisms and offer valuable resources for effective and sustainable control strategies.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Liu X, Zhang L, Zhang N, et al (2024)

CRISPR/Cas9-mediated Nap knockout affects female reproduction and egg shape in Bombyx mori.

Insect molecular biology, 33(6):722-731.

Insect reproductive capacity can affect effective pest control and infertility studies and has become an important focus in recent molecular genetic research. Nucleosome assembly protein (Nap) is highly conserved across multiple species and is involved in forming the sperm nucleus in many species. We used clustered regularly interspaced palindromic repeats/Cas9 technology to knockout BmNap in Bombyx mori and observed that the mutations caused female infertility, whereas male fertility was not affected. BmNap mutants grew and mated normally; however, female mutants laid smaller eggs that could not be fertilised and did not hatch. In addition, female sterility produced by the mutation could be inherited stably via male mutants; therefore, Nap could be used as a potential target for lepidopteran pest control through population regulation. In the current study, we elucidated a new function of BmNap, increased the understanding of the oogenesis regulation network in Lepidoptera and promoted the development of insect sterility technologies.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Wu M, Sun H, Wang A, et al (2024)

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Insect molecular biology, 33(6):732-743.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

RevDate: 2024-11-05
CmpDate: 2024-11-05

Han J, Klobasa W, de Oliveira L, et al (2024)

CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection.

Insect molecular biology, 33(6):589-600.

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-colour genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-colour phenotype. Specifically, while young larvae exhibit orange-coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body colour, with Fo-w mutants having a lighter coloured body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene function and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.

RevDate: 2024-11-04

Pulman J, Botto C, Malki H, et al (2024)

Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina.

Molecular therapy. Nucleic acids, 35(4):102349.

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo. However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and retinal pigment epithelium cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future.

RevDate: 2024-11-04

Mamatha Bhanu LS, Kataki S, S Chatterjee (2024)

CRISPR: New promising biotechnological tool in wastewater treatment.

Journal of microbiological methods pii:S0167-7012(24)00178-7 [Epub ahead of print].

The increasing demand for water resources with increase in population has sparked interest in reusing produced water, especially in water-scarce regions. The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an emerging genome editing tool that has the potential to trigger significant impact with broad application scope in wastewater treatment. We provide a comprehensive overview of the scope of CRISPR-Cas based tool for treating wastewater that may bring new scope in wastewater management in future in controlling harmful contaminants and pathogens. As an advanced versatile genome engineering tool, focusing on particular genes and enzymes that are accountable for pathogen identification, regulation of antibiotic/antimicrobial resistance, and enhancing processes for wastewater bioremediation constitute the primary focal points of research associated with this technology. The technology is highly recommended for targeted mutations to incorporate desirable microalgal characteristics and the development of strains capable of withstanding various wastewater stresses. However, concerns about gene leakage from strains with modified genome and off target mutations should be considered during field application. A comprehensive interdisciplinary approach involving various fields and an intense research focus concerning delivery systems, target genes, detection, environmental conditions, and monitoring at both lab and ground level should be considered to ensure its successful application in sustainable and safe wastewater treatment.

RevDate: 2024-11-03

Liu Y, Bai X, Feng X, et al (2024)

Revolutionizing animal husbandry: Breakthroughs in gene editing delivery systems.

Gene pii:S0378-1119(24)00925-9 [Epub ahead of print].

Gene editing technology has become an essential tool for advancing breeding practices, enhancing disease resistance, and boosting productivity in animal husbandry. Despite its potential, the delivery of gene editing reagents into cells faces several challenges, including low targeting efficiency, immunogenicity, and cytotoxicity, which have hindered its wider application in the field. This review discusses the evolution of gene editing technologies and highlights recent advancements in various delivery methods used in animal husbandry. It critically evaluates the strengths and weaknesses of these different delivery approaches while identifying potential directions for future development. The goal is to equip researchers with effective strategies to optimize delivery methods, ultimately facilitating the implementation and progress of gene editing technologies in animal husbandry.

RevDate: 2024-11-03
CmpDate: 2024-11-03

Mao G, Li Q, Zhang Z, et al (2024)

Analyte-induced hindrance in the RCA-assisted CRISPR/Cas12a system for homogeneous protein assays.

Analytica chimica acta, 1330:343294.

Heterogeneous assays, such as enzyme-linked immunosorbent assays, have become indispensable for in vitro diagnostics. However, the simple, sensitive, and accurate detection is limited by their multiple washing and incubation steps, and limited amplification methods. In this study, we design a novel approach utilizing analyte-induced hindrance within the rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for simple and highly sensitive homogenous protein detection. Streptavidin (SA) and digoxin antibody (anti-Dig) are employed as representative detection models. The specific recognition of target proteins using primers modified with small molecules hinders the RCA process, preventing the activation of Cas12a's trans-cleavage activity, thereby leading to a reduction in fluorescence intensity. Our developed platform exhibites exceptional detection performance characterized by high sensitivity, robust specificity, and significant potential for application in complex samples. By expanding the recognition elements, this platform can evolve into a versatile clinical diagnostic tool with universal applicability. In addition, this platform provides a novel direction for quantifying ultralow-concentration disease biomarkers in clinical practice.

RevDate: 2024-11-03
CmpDate: 2024-11-03

Pei Z, Su Z, Chen J, et al (2024)

A nanopore-based label-free CRISPR/Cas12a system for portable and ultrasensitive detection of zearalenone.

Analytica chimica acta, 1330:343280.

BACKGROUND: Food safety has become a serious global concern. Therefore, there is a need for effective detection technologies in this field. Currently, the development of effective on-site detection techniques is extremely important for food safety. However, the traditional on-site detection methods currently lack effective signal amplification. Herein, the aim of this study was to construct a nanopore-based label-free CRISPR/Cas12a system for the detection of Zearalenone (ZEN). The method is expected to be highly sensitive for portable detection of ZEN in food.

RESULTS: The proposed strategy was mainly involved three steps, including the displacement of the target DNA, the triggering of the cleavage of hairpin DNA probes (probes 1) by the trans-cleavage of CRISPR/Cas12a, and the generation of a measurable nanopore current signal. The probes 1 and DNA after the cleavage of probes 1 (probes 2) produce different characteristic nanopore signals as they pass through the nanopore. The established method achieved a low limit of detection (LOD) of 6.52 fM for ZEN and a wide liner range under optimized conditions. Furthermore, the practical applicability of this method was verified in real maize samples and showed good recoveries (90.68-101.98 %) and low relative standard deviations (RSD) (9.21-9.72 %). Therefore, this method is a promising option for rapid and ultrasensitive detection of ZEN.

SIGNIFICANCE AND NOVELTY: The study presented a portable nanopore-based CRISPR/Cas12a signal amplification detection system for the detection of ZEN in food, which had a low LOD and the advantages of rapid, portability, and on-site detection potential. In conclusion, the method presented a promising prospect and universal platform for the detection of ZEN and other mycotoxins, offering a novel insight into on-site food safety detection.

RevDate: 2024-11-03

Kursheed F, Naza E, Mateena S, et al (2024)

CRISPR applications in microbial World: Assessing the opportunities and challenges.

Gene pii:S0378-1119(24)00956-9 [Epub ahead of print].

Genome editing has emerged during the past few decades in the scientific research area to manipulate genetic composition, obtain desired traits, and deal with biological challenges by exploring genetic traits and their sequences at a level of precision. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing tool has offered a much better understanding of cellular and molecular mechanisms. This technology emerges as one of the most promising candidates for genome editing, offering several advantages over other techniques such as high accuracy and specificity. In the microbial world, CRISPR/Cas technology enables researchers to manipulate the genetic makeup of micro-organisms, allowing them to achieve almost impossible tasks. This technology initially discovered as a bacterial defense mechanism, is now being used for gene cutting and editing to explore more of its dimensions. CRISPR/Cas 9 systems are highly efficient and flexible, leading to its widespread uses in microbial research areas. Although this technology is widely used in the scientific community, many challenges, including off-target activity, low efficiency of Homology Directed Repair (HDR), and ethical considerations, still need to be overcome before it can be widely used. As CRISPR/Cas technology has revolutionized the field of microbiology, this review article aimed to present a comprehensive overview highlighting a brief history, basic mechanisms, and its application in the microbial world along with accessing the opportunities and challenges.

RevDate: 2024-11-04
CmpDate: 2024-11-02

Pudgerd A, Saedan S, Santimanawong W, et al (2024)

Genome editing of WSSV CRISPR/Cas9 and immune activation extends the survival of infected Penaeus vannamei.

Scientific reports, 14(1):26306.

White spot syndrome virus (WSSV) is an exceptionally harmful virus that generally causes high levels of mortality in cultured shrimp. Attempts at viral suppression have been made to control the disease and have achieved limited efficiency. Recent advances in genome editing technology using CRISPR/Cas9 have led to potential innovations to prevent or treat many viral diseases. In this study, a CRISPR/Cas9 system was applied to WSSV genome cleavage to suppress WSSV infection in shrimp. The U6 promoter sequence was identified. A chimeric DNA vector consisting of the shrimp U6 promoter with gRNA expression sequences specific to two sites of the WSSV genome and the WSSV ribonucleotide reductase promoter with the Cas9 DNA sequence in pAC-sgRNA-Cas9 was constructed. The expression of gRNAs specific to the WSSV genome and Cas9 was determined in primary cultured hemocyte cells and in shrimp tissue via RT‒PCR. The efficacy of CRISPR/Cas9-WSSV for WSSV genome cleavage was determined in vitro and against WSSV-infected Penaeus vannamei. The reaction of synthetic gRNAs and recombinant Cas9 was able to cleave WSSV DNA amplicons, and shrimp that received CRISPR/Cas9-WSSV presented significantly lower WSSV DNA. In addition to interfering with viral DNA propagation, CRISPR/Cas9-WSSV encapsulated with IHHNV-VLP also stimulated an immune-related gene response. Treatment with CRISPR/Cas9-WSSV against WSSV challenge resulted in a significantly longer survival period. This finding has led to the development and application of a CRISPR/Cas9 system for WSSV infectious disease control, which could be used for managing shrimp aquaculture in the future.

RevDate: 2024-11-04
CmpDate: 2024-11-03

Zhou Z, Zhi T, Zou J, et al (2024)

Transcriptome analysis to identify genes related to programmed cell death resulted from manipulating of BnaFAH ortholog by CRISPR/Cas9 in Brassica napus.

Scientific reports, 14(1):26389.

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of the tyrosine degradation pathway. In this study, we isolated and characterized two homologous BnaFAH genes in Brassica napus L. variant Westar, and then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with single or double-null bnafah alleles. Among these mutant lines, the aacc (bnafah) double-null mutant line, rather than the aaCC (bnaa06fah) mutant line, exhibited programmed cell death (PCD) under short days (SD). Histochemical staining and content measurement confirmed that the accumulation of reactive oxygen species (ROS) in bnafah was significantly higher than that in bnaa06fah. To further elucidate the mechanism of PCD, we performed transcriptomic analyses of bnaa06fah and bnafah at different SD stages. A heatmap cluster of differentially expressed genes (DEGs) revealed that PCD may be related to various redox regulatory genes involved in antioxidant activity, ROS-responsive regulation and calcium signaling. Combined with the results of previous studies, our work revealed that the expression levels of BnaC04CAT2, BnaA09/C09SAL1, BnaA08/C08ACO2, BnaA07/C06ERO1, BnaA08ACA1, BnaC04BIK1, BnaA09CRK36 and BnaA03CPK4 were significantly different and that these genes might be candidate hub genes for PCD. Together, our results underscore the ability of different PCD phenotypes to alter BnaFAH orthologs through gene editing and further elucidated the molecular mechanisms of oxidative stress-induced PCD in plants.

RevDate: 2024-11-02
CmpDate: 2024-11-02

Semcesen LN, Robinson DRL, DA Stroud (2024)

Generating mammalian knock-out cell lines to investigate mitochondrial protein complex assembly.

Methods in enzymology, 707:441-473.

The development of easy-to-use gene-editing approaches has revolutionized the study of mitochondrial protein complex assembly in mammalian cells. Once the domain of classical cell biology models such Saccharomyces cerevisiae, human knockout cell lines lacking expression of a specific protein can be made at low cost and in as little as two to three weeks. In this chapter we outline our approach to generation of knockouts in commonly used transformed laboratory cell lines, with a view toward their use in the study of mitochondrial respiratory chain complex assembly and mitochondrial biology. Common pitfalls and caveats are discussed along with recommendations on how to validate a knockout cell line through sequencing of genomic edits and stable complementation to exclude the influence of off-target effects and enable further studies of protein function.

RevDate: 2024-11-02
CmpDate: 2024-11-02

Mehlmann LM, Uliasz TF, Yee SP, et al (2024)

Generation and Characterization of a TRIM21 Overexpressing Mouse Line.

Genesis (New York, N.Y. : 2000), 62(5):e23616.

Specific removal of a protein is a key to understanding its function. "Trim-Away" utilizes TRIM21, an antibody receptor and ubiquitin ligase, for acute and specific reduction of proteins. When TRIM21 is expressed in cells, introduction of a specific antibody causes rapid degradation of the targeted protein; however, TRIM21 is endogenously expressed in few cell types. We have generated a mouse line using CRISPR to insert a conditional overexpression cassette of TRIM21 into the safe harbor site, Rosa26. These conditionally-expressing mice can be bred to a wide variety of Cre mice to target cell-specific TRIM21 overexpression in different tissues. Zp3[Cre] mice expressed TRIM21 protein specifically in oocytes, whereas Hprt[Cre] mice expressed the protein globally. When TRIM21-overexpressing oocytes were microinjected with specific antibodies targeting either the IP3 receptor or SNAP23, these proteins were effectively degraded. In addition, cortical neural cells from globally-overexpressing TRIM21 mice showed a dramatic reduction in IP3 receptor protein within hours after electroporation of a specific antibody. These experiments confirm the effectiveness of the Trim-Away method for protein reduction. These mice should make a valuable addition to the broader research community, as a wide range of proteins and cell types can be studied using this method.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Kim S, Choi YJ, Eom H, et al (2024)

Fungal degradation of phenylacetate focusing on CRISPR/Cas9-assisted characterization of two oxidative enzyme genes of Akanthomyces muscarius AM1091.

Microbiological research, 289:127934.

The degradation of phenylacetate (PA) was investigated as a model to explore aromatic compound breakdown in the fungal system. Fungal strains capable of utilizing PA as their sole carbon source were isolated using a minimal solid medium supplemented with 0.5 % PA. Subsequent cultivation in minimum liquid medium revealed that selected fungal strains, including Trametes versicolor TV0876 and TV3295, Paecilomyces hepiali PH4477, and Akanthomyces muscarius AM1091, efficiently removed PA within 24 h. HPLC analysis of culture supernatants from various fungal strains revealed a time-dependent accumulation of 2-hydroxyphenylacetate (2-HPA) and 4-hydroxyphenylacetate (4-HPA), two key major metabolic products primarily found in ascomycetes and basidiomycetes, respectively. This suggests that the first hydroxylation of PA is catalyzed by two distinct hydroxylases, one for each fungal group. Furthermore, fungal species that make 4-HPA also produce phenylethanol (PE), indicating a distinct catabolic mechanism to remove PA by direct reduction of PA to PE. A. muscarius AM1091, identified as the most efficient PA degrader in this study, was studied further to determine the biochemical pathway of PA degradation. RNA-Seq and RT-PCR analyses of AM1091 revealed two oxidative enzyme genes, CYP1 and DIO4, upregulated in the presence of PA. Targeted disruption utilizing preassembled Cas9-gRNA ribonucleoprotein complexes and homologous DNAs harboring the URA3 gene as an auxotrophic marker resulted in the cyp1 and dio4 mutant strains. The cyp1 mutant was incapable of converting PA to 2-HPA, indicating its involvement in the C2 hydroxylation, whereas the dio4 mutant was unable to degrade 2,5-dihydroxyphenylacetate (2,5-DHPA), resulting in the accumulation of 2,5-DHPA. Our findings indicate that A. muscarius AM1091 degrades PA through the activities of CYP1 and DIO4 for the C2 hydroxylation and subsequent ring-opening reactions, respectively.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Ahamed MA, Politza AJ, Liu T, et al (2024)

CRISPR-based strategies for sample-to-answer monkeypox detection: current status and emerging opportunities.

Nanotechnology, 36(4):.

The global health threat posed by the Monkeypox virus (Mpox) requires swift, simple, and accurate detection methods for effective management, emphasizing the growing necessity for decentralized point-of-care (POC) diagnostic solutions. The clustered regularly interspaced short palindromic repeats (CRISPR), initially known for its effective nucleic acid detection abilities, presents itself as an attractive diagnostic strategy. CRISPR offers exceptional sensitivity, single-base specificity, and programmability. Here, we reviewed the latest developments in CRISPR-based POC devices and testing strategies for Mpox detection. We explored the crucial role of genetic sequencing in designing crRNA for CRISPR reaction and understanding Mpox transmission and mutations. Additionally, we showed the integration of CRISPR-Cas12 strategy with pre-amplification and amplification-free methods. Our study also focused on the significant role of Cas12 proteins and the effectiveness of Cas12 coupled with recombinase polymerase amplification (RPA) for Mpox detection. We envision the future prospects and challenges, positioning CRISPR-Cas12-based POC devices as a frontrunner in the next generation of molecular biosensing technologies.

RevDate: 2024-11-04
CmpDate: 2024-11-04

LaFleur MW, D'Andrea JM, Patterson DG, et al (2024)

In Vivo CRISPR Screening Reveals CHD7 as a Positive Regulator of Short-lived Effector Cells.

Journal of immunology (Baltimore, Md. : 1950), 213(10):1528-1541.

CD8+ T cells differentiate into two subpopulations in response to acute viral infection: memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). MPECs and SLECs are epigenetically distinct; however, the epigenetic regulators required for formation of these subpopulations are mostly unknown. In this study, we performed an in vivo CRISPR screen in murine naive CD8+ T cells to identify the epigenetic regulators required for MPEC and SLEC formation, using the acute lymphocytic choriomeningitis virus Armstrong infection model. We identified the ATP-dependent chromatin remodeler CHD7 (chromodomain-helicase DNA-binding protein 7) as a positive regulator of SLEC formation, as knockout (KO) of Chd7 reduced SLECs numerically. In contrast, KO of Chd7 increased the formation of central memory T cells following pathogen clearance yet attenuated memory cell expansion following a rechallenge. These findings establish CHD7 as a novel positive regulator of SLEC and a negative regulator of central memory T cell formation.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Lauerer AM, Caravia XM, Maier LS, et al (2024)

Gene editing in common cardiovascular diseases.

Pharmacology & therapeutics, 263:108720.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, highlighting the high socioeconomic impact. Current treatment strategies like compound-based drugs or surgeries are often limited. On the one hand, systemic administration of substances is frequently associated with adverse side effects; on the other hand, they typically provide only short-time effects requiring daily intake. Thus, new therapeutic approaches and concepts are urgently needed. The advent of CRISPR-Cas9 genome editing offers great promise for the correction of disease-causing hereditary mutations. As such mutations are often very rare, gene editing strategies to correct them are not broadly applicable to many patients. Notably, there is recent evidence that gene editing technology can also be deployed to disrupt common pathogenic signaling cascades in a targeted, specific, and efficient manner, which offers a more generalizable approach. However, several challenges remain to be addressed ranging from the optimization of the editing strategy itself to a suitable delivery strategy up to potential immune responses to the editing components. This review article discusses important CRISPR-Cas9-based gene editing approaches with their advantages and drawbacks and outlines opportunities in their application for treatment of cardiovascular diseases.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Ashok K, Nagaraja Bhargava C, Venkatesh R, et al (2025)

Molecular characterization and CRISPR/Cas9 validation of the precursor of egg yolk protein gene, vitellogenin of Leucinodes orbonalis Guenée (Lepidoptera: Crambidae).

Gene, 933:148925.

Vitellogenin (Vg), a yolk protein precursor, plays an important role in the oocyte development of insects and is an important target of genetic pest management. Vg is synthesized in the fat body, transported through haemolymph and accumulates in developing oocytes. In this regard, the eggplant shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major pest in South and South East Asia and a serious concern for farmers. Therefore, in the present study, we have cloned and characterized Vg from L. orbonalis (LoVg) for further applications. The cloned Vg consisted of 5,370 base pairs encoding 1,790 amino acid residues long protein. Further, sequence alignment revealed that LoVg has three conserved domains: a Vitellogenin N domain (LPD-N), a domain of unknown function protein families (DUF1943), and a von Willebrand factor type D domain (VWD). Using phylogenetic analysis, it was found that LoVg evolved alongside homologous proteins from different insects. The real-time expression levels of LoVg were significantly greater in female adults followed by the pupal stage. This suggests that Vg production and absorption in L. orbonalis occurs in the later pupal stage. Our studies showed that editing LoVg using CRISPR/Cas9 did not affect the total number of eggs laid but affected egg hatchability. These studies help us to design newer approaches in insect pest management through genetic suppression for sustainable pest management.

RevDate: 2024-11-01
CmpDate: 2024-11-01

Yuan J, Wang L, He F, et al (2025)

Hue-change coupled with CRISPR-Cas12a lateral flow assay for the semiquantitative detection of Salmo salar adulteration.

Food chemistry, 463(Pt 1):141088.

Salmo salar is one of the most popular salmon species due to its meaty texture and quality protein. Oncorhynchus mykiss, which has a muscle texture similar to that of Salmo salar and is less expensive, is often used as a substitute for Salmo salar. As Salmo salar and Oncorhynchus mykiss belong to the same subfamily of Salmonidae, traditional methods are ineffective in the specific detection of the two. In this study, we combined hue-change with CRISPR/Cas12a lateral flow assay to detect the Salmo salar adulteration. This method detected S. salar genomic DNA at a vLOD of 5 copies, and was able to accurately identify adulterated samples containing 5 % w/w Salmo salar within one hour. In addition, the detection of Salmo salar in processed food products was achieved with the naked-eye at a concentration range of 0 % ∼ 70 % w/w, and the detection accuracy is between 93.3 % ∼ 100 %.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Welikala MU, Butterworth LJ, Behrmann MS, et al (2024)

Tau-mediated coupling between Pol III synthesis and DnaB helicase unwinding helps maintain genomic stability.

The Journal of biological chemistry, 300(10):107726.

The τ-subunit of the clamp loader complex physically interacts with both the DnaB helicase and the polymerase III (Pol III) core α-subunit through domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the dnaX gene at the C terminus of τ predicted to interact with the α-subunit of Pol III. Perturbation of the α-τ binding interaction in vivo resulted in cellular and genomic stress markers that included reduced growth rates, fitness, and viabilities. Specifically, dnaX:mut strains showed increased cell filamentation, mutagenesis frequencies, and activated SOS. In situ fluorescence flow cytometry and microscopy quantified large increases in the amount of ssDNA gaps present. Removal of the C terminus of τ (I618X) still maintained its interactions with DnaB and stimulated unwinding but lost its interaction with Pol III, resulting in significantly reduced rolling circle DNA synthesis. Intriguingly, dnaX:L635P/D636G had the largest induction of SOS, high mutagenesis, and the most prominent ssDNA gaps, which can be explained by an impaired ability to regulate the unwinding speed of DnaB resulting in a faster rate of in vitro rolling circle DNA replication, inducing replisome decoupling. Therefore, τ-stimulated DnaB unwinding and physical coupling with Pol III acts to enforce replisome plasticity to maintain an efficient rate of synthesis and prevent genomic instability.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Lewis MW, King CM, Wisniewska K, et al (2024)

CRISPR Screening of Transcribed Super-Enhancers Identifies Drivers of Triple-Negative Breast Cancer Progression.

Cancer research, 84(21):3684-3700.

Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer, which is due in part to the paucity of targeted therapies. A systematic analysis of regulatory elements that extend beyond protein-coding genes could uncover avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their noncoding enhancer RNA (eRNA) transcripts. The functions of the top 30 eRNA-producing super-enhancers were systematically probed using high-throughput CRISPR-interference assays coupled to RNA sequencing that enabled unbiased detection of target genes genome-wide. Generation of high-resolution Hi-C chromatin interaction maps enabled annotation of the direct target genes for each super-enhancer, which highlighted their proclivity for genes that portend worse clinical outcomes in patients with TNBC. Illustrating the utility of this dataset, deletion of an identified super-enhancer controlling the nearby PODXL gene or specific degradation of its eRNAs led to profound inhibitory effects on target gene expression, cell proliferation, and migration. Furthermore, loss of this super-enhancer suppressed tumor growth and metastasis in TNBC mouse xenograft models. Single-cell RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses demonstrated the enhanced activity of this super-enhancer within the malignant cells of TNBC tumor specimens compared with nonmalignant cell types. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of TNBC. Significance: Integrative analysis of eRNA-producing super-enhancers defines molecular mechanisms controlling global patterns of gene expression that regulate clinical outcomes in breast cancer, highlighting the potential of enhancers as biomarkers and therapeutic targets.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Cai Y, Liang Y, Shi H, et al (2024)

Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR-mediated disruption of Transparent Testa 8.

Plant biotechnology journal, 22(10):2773-2784.

Camelina (Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Wang M, Jiang N, Xu Y, et al (2024)

CmBr confers fruit bitterness under CPPU treatment in melon.

Plant biotechnology journal, 22(10):2724-2737.

Many biotic or abiotic factors such as CPPU (N-(2-chloro-pyridin-4-yl)-N'-phenylurea), a growth regulator of numerous crops, can induce bitterness in cucurbits. In melon, cucurbitacin B is the major compound leading to bitterness. However, the molecular mechanism underlying CuB biosynthesis in response to different conditions remains unclear. Here, we identified a set of genes involved in CPPU-induced CuB biosynthesis in melon fruit and proposed CmBr gene as the major regulator. Using CRISPR/Cas9 gene editing, we confirmed CmBr's role in regulating CuB biosynthesis under CPPU treatment. We further discovered a CPPU-induced MYB-related transcription factor, CmRSM1, which specifically binds to the Myb motif within the CmBr promoter and activates its expression. Moreover, we developed an introgression line by introducing the mutated Cmbr gene into an elite variety and eliminated CPPU-induced bitterness, demonstrating its potential application in breeding. This study offers a valuable tool for breeding high-quality non-bitter melon varieties and provides new insights into the regulation of secondary metabolites under environmental stresses.

RevDate: 2024-11-04
CmpDate: 2024-11-04

Brant EJ, Eid A, Kannan B, et al (2024)

The extent of multiallelic, co-editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield.

Plant biotechnology journal, 22(10):2660-2671.

Sugarcane (Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 (LG1) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9-mediated co-mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2n = 100-120, x = 10-12). Next generation sequencing revealed co-editing frequencies of 7.4%-100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co-editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi-row field plots. The co-edited LG1 mutations were stably maintained in vegetative progenies and the extent of co-editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co-editing of LG1 in highly polyploid sugarcane allows fine-tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

RevDate: 2024-11-01

Liu X, Ji P, Liao J, et al (2024)

CRISPR/Cas knockout of the NADPH oxidase gene OsRbohB reduces ROS overaccumulation and enhances heat stress tolerance in rice.

Plant biotechnology journal [Epub ahead of print].

Heat stress (HS) has become a major factor limiting crop yields worldwide. HS inhibits plant growth by ROS accumulation, and NADPH oxidases (Rbohs) are major ROS producers in plants. Here, we show that CRISPR/Cas knockout of the OsRbohB (OsRbohB-KO) significantly increased rice tolerance to HS imposed at various different growth stages. We produced OsRbohB-KO and OsRbohB-overexpression (OsRbohB-OE) lines in a japonica cultivar, Nipponbare. Compared with nontransgenic wild-type (WT) plants, the OsRbohB-KO lines showed a significant increase in chlorophyll contents (5.2%-58.0%), plant growth (48.2%-65.6%) and grain yield (8.9%-20.5%), while reducing HS-induced ROS accumulation in seeds (21.3%-33.0%), seedlings (13.0%-30.4%), anthers (13.1%-20.3%) and grains (9.7%-22.1%), under HS conditions. Analysis of yield components revealed that the increased yield of OsRbohB-KO plants was due to increased starch synthetase activity, spikelets per panicle (2.0%-9.3%), filled spikelets (4.8%-15.5%), percentage of filled spikelets (2.4%-6.8%) and 1000-grain weight (2.9%-7.4%) under HS conditions during the reproductive stage. Grain milling and appearance quality, and starch content were also significantly increased in OsRbohB-KO plants under HS conditions during the mature stage. Furthermore, OsRbohB-KO significantly upregulated the expression levels of heat shock-related genes, OsHSP23.7, OsHSP17.7, OsHSF7 and OsHsfA2a, in rice seedlings and grains under long-term HS conditions. Conversely, OsRbohB-OE resulted in phenotypes that were opposite to OsRbohB-KO in most cases. Our results suggest that suppression of OsRbohB provides an effective approach for alleviating heat damage and improving grain yield and quality of rice under long-term HS conditions.

RevDate: 2024-11-01
CmpDate: 2024-11-01

Gilmore RB, Gorka D, Stoddard CE, et al (2024)

Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cells.

PloS one, 19(11):e0311565.

Angelman syndrome (AS) and Prader-Willi syndrome (PWS), two distinct neurodevelopmental disorders, result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele, respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless, gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences, reprogramming methods, and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here, we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally, these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS.

RevDate: 2024-11-01

Margolis SR, AJ Meeske (2024)

Crosstalk between three CRISPR-Cas types enables primed type VI-A adaptation in Listeria seeligeri.

bioRxiv : the preprint server for biology pii:2024.10.25.620265.

CRISPR-Cas systems confer adaptive immunity to their prokaryotic hosts through the process of adaptation, where sequences are captured from foreign nucleic acids and integrated as spacers in the CRISPR array, and thereby enable crRNA-guided interference against new threats. While the Cas1-2 integrase is critical for adaptation, it is absent from many CRISPR-Cas loci, rendering the mechanism of spacer acquisition unclear for these systems. Here we show that the RNA-targeting type VI-A CRISPR system of Listeria seeligeri acquires spacers from DNA substrates through the action of a promiscuous Cas1-2 integrase encoded by a co-occurring type II-C system, in a transcription-independent manner. We further demonstrate that the type II-C integration complex is strongly stimulated by preexisting spacers in a third CRISPR system (type I-B) which imperfectly match phage targets and prime type VI-A adaptation. Altogether, our results reveal an unprecedented degree of communication among CRISPR-Cas loci encoded by a single organism.

RevDate: 2024-11-02
CmpDate: 2024-11-01

Chege Kuria T, Schneider A, Baraka F, et al (2024)

In vivo analysis of CRISPR-edited germinal center murine B cells.

Frontiers in immunology, 15:1473760.

The germinal center (GC) reaction is crucial for somatic hypermutation, affinity maturation, and the selection of high-affinity B cells, all of which are hallmarks of the humoral immune response. Understanding the distinct roles of various B cell genes is essential for elucidating the selection mechanisms within the GC reaction. Traditionally, studying B cell gene function in the GC reaction involved generating knock-out mice, a highly time-consuming method that necessitates complex vectors. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has simplified the creation of knock-out mice. However, even with CRISPR, the generation of knock-out mice still faces challenges, including being time-consuming, costly, having low knock-out efficiency, and raising ethical concerns regarding animal use. To address these challenges, we developed an alternative method to traditional knock-out mouse generation. Our approach entails the ex vivo CRISPR editing of B cells from transgenic donor mice with different B cell receptor affinities followed by their adoptive transfer into recipient mice. We present a cost-effective, rapid, versatile, and adaptable CRISPR-Cas9 method for in vivo loss-of-function studies of individual murine B cell genes within the context of the GC reaction. This method provides a valuable tool for investigating the complex roles of different B cell genes in the GC selection process. As proof of concept, we validated our approach by examining the role of the pro-apoptotic gene Fas in the GC selection process. We adoptively transferred a mix of Fas knock-out (Fas[KO]) low-affinity B cells, Fas wild-type (Fas[WT]) low-affinity B cells, and Fas[WT] high-affinity B cells into recipient mice. From our results, Fas[KO] low-affinity B cells were still outcompeted by the Fas[WT] high-affinity B cells for selection in the GC. An important observation was the accumulation of Fas[KO] low-affinity GC B cells when compared to the Fas[WT] low-affinity B cells, which suggested a role of Fas in the GC selection process.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Amoah P, Oumarou Mahamane AR, Byiringiro MH, et al (2024)

Genome editing in Sub-Saharan Africa: a game-changing strategy for climate change mitigation and sustainable agriculture.

GM crops & food, 15(1):279-302.

Sub-Saharan Africa's agricultural sector faces a multifaceted challenge due to climate change consisting of high temperatures, changing precipitation trends, alongside intensified pest and disease outbreaks. Conventional plant breeding methods have historically contributed to yield gains in Africa, and the intensifying demand for food security outpaces these improvements due to a confluence of factors, including rising urbanization, improved living standards, and population growth. To address escalating food demands amidst urbanization, rising living standards, and population growth, a paradigm shift toward more sustainable and innovative crop improvement strategies is imperative. Genome editing technologies offer a promising avenue for achieving sustained yield increases while bolstering resilience against escalating biotic and abiotic stresses associated with climate change. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) is unique due to its ubiquity, efficacy, alongside precision, making it a pivotal tool for Sub-Saharan African crop improvement. This review highlights the challenges and explores the prospect of gene editing to secure the region's future foods.

RevDate: 2024-11-01
CmpDate: 2024-11-01

Kim MC, Gate R, Lee DS, et al (2024)

Method of moments framework for differential expression analysis of single-cell RNA sequencing data.

Cell, 187(22):6393-6410.e16.

Differential expression analysis of single-cell RNA sequencing (scRNA-seq) data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remain challenging. We introduce Memento, a tool for robust and efficient differential analysis of mean expression, variability, and gene correlation from scRNA-seq data, scalable to millions of cells and thousands of samples. We applied Memento to 70,000 tracheal epithelial cells to identify interferon-responsive genes, 160,000 CRISPR-Cas9 perturbed T cells to reconstruct gene-regulatory networks, 1.2 million peripheral blood mononuclear cells (PBMCs) to map cell-type-specific quantitative trait loci (QTLs), and the 50-million-cell CELLxGENE Discover corpus to compare arbitrary cell groups. In all cases, Memento identified more significant and reproducible differences in mean expression compared with existing methods. It also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by perturbations.

RevDate: 2024-11-02
CmpDate: 2024-11-01

Liu H, Zhao XF, Lu YN, et al (2024)

CRISPR/Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA.

The Journal of clinical investigation, 134(21):.

A hexanucleotide GGGGCC repeat expansion in the non-coding region of the C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR/Cas13d, a powerful RNA-targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR/Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR/Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR/Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.

RevDate: 2024-10-31

Liu X, Dong H, Wang H, et al (2024)

Recent Advances in Genetic Engineering Strategies of Sinorhizobium meliloti.

ACS synthetic biology [Epub ahead of print].

Sinorhizobium meliloti is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. S. meliloti has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B12. Advances in gene editing tools play a vital role in the development of S. meliloti strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in S. meliloti. This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in S. meliloti are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of S. meliloti, and serve as a reference for the utilization of genome editing tools for other Rhizobium species.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Wang X, Hua DN, Zhou J, et al (2024)

[Effect of Tumor Suppressor Gene Kmt2c Heterozygous Deletion on Hematopoietic System in Mice].

Zhongguo shi yan xue ye xue za zhi, 32(5):1571-1577.

OBJECTIVE: To explore the effect of heterozygous deletion of histone methyltransferase Kmt2c gene on the hematological system of mice.

METHODS: CRISPR/Cas9 technology was used to construct mice model of Kmt2c heterozygous deletion (Kmt2c[+/-]) and the changes of whole blood cell count in mice were continuously monitored by blood routine test. The clonal expansion ability of bone marrow cells was explored by colony formation assay in vitro and the proportion of primitive hematopoietic cells, including long-term hematopoietic stem cell (LT-HSC), short-term hematopoietic stem cell (ST-HSC), and multipotent progenitor cell in mutant mice was analyzed by flow cytometry.

RESULTS: Kmt2c[+/-] mice model was successfully constructed, and the mRNA expression level of Kmt2c was 28% of that of C57BL/6J mice. The colony formation ability of bone marrow cells of Kmt2c[+/-] mice in vitro increased with the passage times, and the colony number in the fourth generation was significantly higher than that of control group (P <0.05). The proportions of LT-HSC and ST-HSC in the primitive hematopoietic cell population of Kmt2c[+/-] mice was 19.6%±3.3% and 28.9%±4.9%, respectively, which showed an increasing trend compared with 16.9%±2.6% and 18.9%±2.5% in control group, but the difference was not statistically significant (P >0.05). The white blood cell count of Kmt2c[+/-] mice gradually increased after 12 weeks of monitoring and reached (9.8±1.0)×10[9]/L at the 14[th] week, which was significantly higher than (7.3±1.4)×10[9]/L of control group (P < 0.05).

CONCLUSION: The bone marrow cells of Kmt2c[+/-] mice have potential of clonal expansion.

RevDate: 2024-10-31

Pérez AA, Vazquez-Meves G, ME Hunter (2024)

Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods.

The CRISPR journal [Epub ahead of print].

Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is a critical component to mitigate the impact of animal diseases in these sectors. To monitor human diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable of detecting unique DNA and RNA sequences related to their associated pathogens. However, despite the significant advances in the general development of CRISPR-Cas biosensors, their use to support wildlife disease management is lagging. In some cases, wildlife diseases of concern could be rapidly surveyed using these tools with minimal technical, operational, or cost requirements to end users. This review explores the potential to further leverage this technology to advance wildlife disease monitoring and highlights how concerted standardization of protocols can help to ensure data reliability.

RevDate: 2024-10-31

Uranga M, Martín-Hernández AM, De Storme N, et al (2024)

CRISPR-Cas systems and applications for crop bioengineering.

Frontiers in bioengineering and biotechnology, 12:1483857.

CRISPR-Cas technologies contribute to enhancing our understanding of plant gene functions, and to the precise breeding of crop traits. Here, we review the latest progress in plant genome editing, focusing on emerging CRISPR-Cas systems, DNA-free delivery methods, and advanced editing approaches. By illustrating CRISPR-Cas applications for improving crop performance and food quality, we highlight the potential of genome-edited crops to contribute to sustainable agriculture and food security.

RevDate: 2024-10-31

Yuan G, Deng S, Czajka JJ, et al (2024)

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger.

Frontiers in bioengineering and biotechnology, 12:1452496.

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Islam M, Yang Y, Simmons AJ, et al (2024)

Temporal recording of mammalian development and precancer.

Nature, 634(8036):1187-1195.

Temporal ordering of cellular events offers fundamental insights into biological phenomena. Although this is traditionally achieved through continuous direct observations[1,2], an alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible marks that enables retrospective temporal ordering[3-5]. Using a multipurpose, single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, with incorporation of cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during mouse embryonic development, unconventional developmental relationships between cell types and new epithelial progenitor states by their unique genetic histories. Analysis of mouse adenomas, coupled to multiomic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyclonal initiation in 15-30% of colonic precancers, showing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses, to explore the origins and timing of development and tumorigenesis in mammalian systems.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Singpanomchai N, P Ratthawongjirakul (2024)

The CRISPR-dCas9 interference system suppresses inhA gene expression in Mycobacterium smegmatis.

Scientific reports, 14(1):26116.

CRISPR-dead Cas9 interference (CRISPRi) has become a valuable tool for precise gene regulation. In this study, CRISPRi was designed to target the inhA gene of Mycobacterium smegmatis (Msm), a gene necessary for mycolic acid synthesis. Our findings revealed that sgRNA2 induced with 100 ng/ml aTc achieved over 90% downregulation of inhA gene expression and inhibited bacterial viability by approximately 1,000-fold. Furthermore, CRISPRi enhanced the susceptibility of M. smegmatis to isoniazid and rifampicin, which are both 50% and 90% lower than those of the wild-type strain or other strains, respectively. This study highlights the ability of CRISPRi to silence the inhA gene, which impacts bacterial viability and drug susceptibility. The findings provide valuable insights into the utility of CRISPRi as an alternative tool for gene regulation. CRISPRi might be further assessed for its synergistic effect with current anti-tuberculosis drugs and its possible implications for combating mycobacterial infections, especially drug-resistant tuberculosis.

RevDate: 2024-10-30

Gogoi N, Susila H, Leach J, et al (2024)

Developing frameworks for nanotechnology-driven DNA-free plant genome-editing.

Trends in plant science pii:S1360-1385(24)00264-4 [Epub ahead of print].

The bottlenecks of conventional plant genome-editing methods gave an innovative rise to nanotechnology as a delivery tool to manipulate gene(s) of interest. Studies suggest a strong correlation between the physicochemical properties of nanomaterials and their efficiency in gene delivery to different plant species/tissues. In this opinion article we highlight the need for a deeper understanding of plant-nanomaterial interactions to align their full capabilities with the strategic goals of plant genome-editing. Additionally, we emphasize DNA-free plant genome-editing approaches to potentially mitigate concerns surrounding genetically modified organisms (GMOs). Lastly, we propose a strategic integration of the principles of responsible research and innovation (RRI) in R&D. We aim to initiate a dialogue on developing collaborative and socio-technical frameworks for nanotechnology and DNA-free plant genome-editing.

RevDate: 2024-10-30

Li Z, Su X, Lin Y, et al (2024)

Expanding the cell quantity of CRISPR/Cas9 gene editing by continuous microfluidic electroporation chip.

Bioelectrochemistry (Amsterdam, Netherlands), 161:108840 pii:S1567-5394(24)00202-0 [Epub ahead of print].

CRISPR/Cas9-mediated gene editing offers promising and safe therapeutic options for a wide range of diseases. The technical difficulty of efficiently acquiring large quantities of gene-edited therapeutic cells in a short time period is now preventing the widespread clinical application of CRISPR/Cas9-mediated gene editing. Herein, a Large Volume Continuous Electroporation Chip (LaViE-Chip) has been developed to address the challenge of acquiring sufficient quantities of genetically edited cells for CRISPR/Cas9 gene editing. By connecting multiple relatively narrow microfluidic channels in parallel, a satisfactory balance between cell flow volume and electric field uniformity was achieved with two simple off-chip electrodes, which also isolated harmful effects around electrodes from target cells. Meanwhile, by carefully designing the curvature of the microfluidic channel, hydrodynamic controlled rotation of target cells has been realized to improve the transfection efficiency and cell viability. With these improvements, the LaViE-Chip realized 71.06 % electrotransfection efficiency, 84.3 % cell viability, and 10[7] cell/min cell processing speed. Moreover, the first successful incessant CRISPR gene editing by electroporation has been demonstrated, laying the technical foundation of therapeutic CRISPR gene editing.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Aboelhassan DM, H Abozaid (2024)

Opportunities for CRISPR-Cas9 application in farm animal genetic improvement.

Molecular biology reports, 51(1):1108.

CRISPR-Cas9 has emerged as a powerful tool in livestock breeding, enabling precise genetic modifications to address genetic diseases, enhance productivity, and develop disease-resistant animal breeds. A thorough analysis of previous research highlights the potential of CRISPR-Cas9 in overcoming genetic disorders by targeting specific mutations in genes. Furthermore, its integration with reproductive biotechnologies and genomic selection facilitates the production of gene-edited animals with high genomic value, contributing to genetic enhancement and improved productivity. Additionally, CRISPR-Cas9 opens new avenues for developing disease-resistant livestock and creating innovative breeding models for high-quality production. A key trend in the field is the development of multi-sgRNA vectors to correct mutations in various genes linked to productivity traits or certain diseases within individual genomes, thereby increasing resistance in animals. However, despite the potential advantages of CRISPR-Cas9, public acceptance of genetically modified agricultural products remains uncertain. Would consumers be willing to purchase such products? It is essential to advocate for bold and innovative research into genetically edited animals, with a focus on safety, careful promotion, and strict regulatory oversight to align with long-term goals and public acceptance. Continued advancements in this technology and its underlying mechanisms promise to improve poultry products and genetically modified livestock. Overall, CRISPR-Cas9 technology offers a promising pathway for advancing livestock breeding practices, with opportunities for genetic improvement, enhanced disease resistance, and greater productivity.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Vinhal ALO, de Araújo MRB, Rodrigues EB, et al (2024)

First comparative genomics analysis of Corynebacterium auriscanis.

Memorias do Instituto Oswaldo Cruz, 119:e240156.

BACKGROUND: Corynebacterium auriscanis is a bacterial species frequently isolated from dogs with external otitis or dermatitis and a zoonotic pathogen transmitted by dog bite. It is considered an opportunistic pathogen, but its pathogenicity mechanisms are poorly studied. Comparative genomics can identify virulence and niche factors that could contribute to understanding its lifestyle.

OBJECTIVES: The objectives of this project was to compare genomes of C. auriscanis to identify genes related to its virulence and lifestyle.

METHODS: The genome of strain 32 was sequenced using Illumina HiSeq 2500 (Illumina, CA, USA) and assembled using Unicycler. The two other non-redundant genomes from the same species available in GenBank were included in the analysis. All genomes were annotated and checked for taxonomy, assembly quality, mobile elements, CRISPR-Cas systems, and virulence and antimicrobial resistance genes. The virulence genes in the three genomes were compared to the ones from other pathogens commonly isolated with C. auriscanis.

FINDINGS: The species has 42 virulence factors that can be classified as niche factors, due to the absence of true virulence factors found in primary pathogens. The gene rbpA could confer basal levels of resistance to rifampin.

MAIN CONCLUSIONS: The absence of true virulence factors in the three genomes suggests C. auriscanis has an opportunistic pathogen lifestyle.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Ruetz TJ, Pogson AN, Kashiwagi CM, et al (2024)

CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.

Nature, 634(8036):1150-1159.

Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing[1-4]. Several genetic interventions have been found to ameliorate old brain function[5-8], but systematic functional testing of genes in old NSCs-and more generally in old cells-has not been done. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR-Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

RevDate: 2024-10-31
CmpDate: 2024-10-31

Herms A, Fernandez-Antoran D, Alcolea MP, et al (2024)

Self-sustaining long-term 3D epithelioid cultures reveal drivers of clonal expansion in esophageal epithelium.

Nature genetics, 56(10):2158-2173.

Aging epithelia are colonized by somatic mutations, which are subjected to selection influenced by intrinsic and extrinsic factors. The lack of suitable culture systems has slowed the study of this and other long-term biological processes. Here, we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least 1 year, maintaining a three-dimensional structure with proliferative basal cells that differentiate into suprabasal cells, which eventually shed and retain genomic stability. Live imaging over 5 months showed that epithelioids replicate in vivo cell dynamics. Epithelioids support genetic manipulation and enable the study of mutant cell competition and selection in three-dimensional epithelia, and show how anti-cancer treatments modulate competition between transformed and wild-type cells. Finally, a targeted CRISPR-Cas9 screen shows that epithelioids recapitulate mutant gene selection in aging human esophagus and identifies additional drivers of clonal expansion, resolving the genetic networks underpinning competitive fitness.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Oh S, Santiago G, Manjunath L, et al (2024)

A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection.

Proceedings of the National Academy of Sciences of the United States of America, 121(45):e2412725121.

OAS-RNase L is a double-stranded RNA-induced antiviral pathway triggered in response to diverse viral infections. Upon activation, OAS-RNase L suppresses virus replication by promoting the decay of host and viral RNAs and inducing translational shutdown. However, whether OASs and RNase L are the only factors involved in this pathway remains unclear. Here, we develop CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identifies IRF2 as a key regulator of OAS3. Mechanistically, we demonstrate that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 works in concert with the interferon response through STAT2 to further enhance OAS3 expression. We propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense. This rapid response provides host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Kumar S, Hsiao YW, Wong VHY, et al (2024)

Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina.

Proceedings of the National Academy of Sciences of the United States of America, 121(45):e2408345121.

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a transient manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression of various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence VEGFA mRNA in human retinal organoids and humanized VEGF transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 by AAV2.7m8 can efficiently transduce mouse retinal cells for specific knockdown of human VEGFA in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity, and establish the Cas13bt3 RNA editing system as a potential anti-VEGF agent that can achieve significant control of VEGFA for the treatment of retinal neovascularization.

RevDate: 2024-10-29

Baca CF, Majumder P, Hickling JH, et al (2024)

The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.

Cell pii:S0092-8674(24)01150-4 [Epub ahead of print].

Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cAn) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA4 or cA6 to its CARF domain, Cad1 converts ATP to ITP, both in vivo and in vitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cAn during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.

RevDate: 2024-10-30

Heshmatpour N, Kazemi SM, Schmidt ND, et al (2024)

Targeting DLBCL by mutation-specific disruption of cancer-driving oncogenes.

Frontiers in genome editing, 6:1427322.

Diffuse large B cell lymphomas (DLBCL) are highly aggressive tumors. Their genetic complexity and heterogeneity have hampered the development of novel approaches for precision medicine. Our study aimed to develop a personalized therapy for DLBCL by utilizing the CRISPR/Cas system to induce knockouts (KO) of driver genes, thereby causing cancer cell death while minimizing side effects. We focused on OCI-LY3 cells, modeling DLBCL, and compared them with BJAB cells as controls. Analysis of whole exome sequencing revealed significant mutations in genes like PAX5, CD79B, and MYC in OCI-LY3 cells. CRISPR/Cas9-mediated KO of these genes resulted in reduced cancer cell viability. Subsequent single and dual gRNA targeting of PAX5 mutations inhibited proliferation specifically in OCI-LY3 cells. Moreover, dual gRNA targeting of PAX5 and MYC induced chromosomal rearrangements, reducing cell proliferation substantially. However, targeting single intronic mutations did not affect cell viability, highlighting the importance of disrupting protein function. Targeting multiple mutations simultaneously addresses intra-tumoral heterogeneity, and the transient delivery of CRISPR/Cas9 allows for permanent gene disruption. While challenges such as incomplete editing efficiency and delivery limitations exist, further optimization may enhance therapeutic efficacy. Overall, our findings demonstrate the efficacy of CRISPR/Cas9 in targeting oncogenic mutations, opening avenues for precision medicine in DLBCL treatment.

RevDate: 2024-10-30

Awan MJA, Amin I, Rasheed A, et al (2024)

Knockout mutation in TaD27 enhances number of productive tillers in hexaploid wheat.

Frontiers in genome editing, 6:1455761.

Recent advances allow the deployment of cluster regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases (Cas) system for the targeted mutagenesis in the genome with accuracy and precision for trait improvement in crops. CRISPR-Cas systems have been extensively utilized to induce knockout or frameshift mutations in the targeted sequence of mostly negative regulating genes for wheat improvement. However, most of the reported work has been done in non-commercial varieties of wheat and introgression of edited alleles into breeding population comes with the penalty of unwanted linkage-drag. Wheat yield is controlled by various genes such as positive and negative regulators. The TaD27 gene is described as a negative regulator of shoot branching or tillering and involved in the biosynthesis of strigolactones. In this study, we developed Tad27 knockout mutant lines of an elite wheat cultivar that showed a twofold increase in the number of tillers and 1.8-fold increase in the number of grains per plant. Subsequently, enhancing the grain yield without any morphological penalty in the architecture of the plants. The co-transformation of regeneration enhancing growth regulator, Growth Regulating Factor 4 (GRF4) and its cofactor GRF-Interacting Factor 1 (GIF1), under single T-DNA cassette improved the regeneration efficiency up to 6% of transgenic events from mature embryos of wheat. Our results indicate that the CRISPR-mediated targeted mutagenesis confers the potential to knockout yield-related negative regulators in elite cultivars of wheat that can substantially enhance grain yield per plant and this strategy can be harnessed for the improvement of future wheat.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Čepaitė R, Klein N, Mikšys A, et al (2024)

Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference.

Nature communications, 15(1):9306.

CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Itell HL, Guenthoer J, Humes D, et al (2024)

Host cell glycosylation selects for infection with CCR5- versus CXCR4-tropic HIV-1.

Nature microbiology, 9(11):2985-2996.

Human immunodeficiency virus type 1 (HIV-1) infection involves a selection bottleneck that leads to transmission of one or a few variants. C-C motif chemokine receptor 5 (CCR5) or C-X-C motif chemokine receptor 4 (CXCR4) can act as coreceptors for HIV-1 viral entry. However, initial infection mostly occurs via CCR5, despite abundant expression of CXCR4 on target cells. The host factors that influence HIV-1 susceptibility and selection during transmission are unclear. Here we conduct CRISPR-Cas9 screens and identify SLC35A2 (a transporter of UDP-galactose expressed in target cells in blood and mucosa) as a potent and specific CXCR4-tropic restriction factor in primary target CD4[+] T cells. SLC35A2 inactivation, which resulted in truncated glycans, not only increased CXCR4-tropic infection levels but also decreased those of CCR5-tropic strains consistently. Single-cycle infections demonstrated that the effect is cell-intrinsic. These data support a role for a host protein that influences glycan structure in regulating HIV-1 infection. Host cell glycosylation may, therefore, affect HIV-1 selection during transmission in vivo.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Ferrarese L, Koch M, Baumann A, et al (2024)

Inflammatory Mediators Suppress FGFR2 Expression in Human Keratinocytes to Promote Inflammation.

Molecular and cellular biology, 44(11):489-504.

Fibroblast growth factors (FGFs) are key orchestrators of development, tissue homeostasis and repair. FGF receptor (FGFR) deficiency in mouse keratinocytes causes an inflammatory skin phenotype with similarities to atopic dermatitis, but the human relevance is unclear. Therefore, we generated human keratinocytes with a CRISPR/Cas9-induced knockout of FGFR2. Loss of this receptor promoted the expression of interferon-stimulated genes and pro-inflammatory cytokines under homeostatic conditions and in particular in response to different inflammatory mediators. Expression of FGFR2 itself was strongly downregulated in cultured human keratinocytes exposed to various pro-inflammatory stimuli. This is relevant in vivo, because bioinformatics analysis of bulk and single-cell RNA-seq data showed strongly reduced expression of FGFR2 in lesional skin of atopic dermatitis patients, which likely aggravates the inflammatory phenotype. These results reveal a key function of FGFR2 in human keratinocytes in the suppression of inflammation and suggest a role of FGFR2 downregulation in the pathogenesis of atopic dermatitis and possibly other inflammatory diseases.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Haryani Y, Abdul Halid N, Goh SG, et al (2024)

Efficient metabolic pathway modification in various strains of lactic acid bacteria using CRISPR/Cas9 system for elevated synthesis of antimicrobial compounds.

Journal of biotechnology, 395:53-63.

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Potlapalli BP, Fuchs J, Rutten T, et al (2024)

The potential of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit.

Journal of experimental botany, 75(20):6244-6257.

Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. A recently developed CRISPR-dCas9-based genome labeling tool, known as CRISPR-FISH, allows efficient labeling of repetitive sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR-FISH eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Here, we report on further development of the CRISPR-FISH method, which has been enhanced for increased efficiency through the engineering of a recombinant dCas9 protein containing an ALFA-tag. Using an ALFA-tagged dCas9 protein assembled with an Arabidopsis centromere-specific guide RNA, we demonstrate target-specific labeling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labeled anti-rabbit secondary antibody, resulted in enhanced target-specific signals. The dCas9-ALFA-tag system was also instrumental in live cell imaging of telomeres in Nicotiana benthamiana. This method will further expand the CRISPR imaging toolkit, facilitating a better understanding of genome organization. Furthermore, we report the successful integration of the highly sensitive tyramide signal amplification method with CRISPR-FISH, demonstrating effective labeling of Arabidopsis centromeres.

RevDate: 2024-10-30
CmpDate: 2024-10-30

Wei J, Liu J, Tian Y, et al (2024)

Discovery and engineering of ChCas12b for precise genome editing.

Science bulletin, 69(20):3260-3271.

Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Shen S, Wang W, Ma Y, et al (2024)

Affinity molecular assay for detecting Candida albicans using chitin affinity and RPA-CRISPR/Cas12a.

Nature communications, 15(1):9304.

Invasive fungal infections (IFIs) pose a significant threat to immunocompromised individuals, leading to considerable morbidity and mortality. Prompt and accurate diagnosis is essential for effective treatment. Here we develop a rapid molecular diagnostic method that involves three steps: fungal enrichment using affinity-magnetic separation (AMS), genomic DNA extraction with silicon hydroxyl magnetic beads, and detection through a one-pot system. This method, optimized to detect 30 CFU/mL of C. albicans in blood and bronchoalveolar lavage (BAL) samples within 2.5 h, is approximately 100 times more sensitive than microscopy-based staining. Initial validation using clinical samples showed 93.93% sensitivity, 100% specificity, and high predictive values, while simulated tests demonstrated 95% sensitivity and 100% specificity. This cost-effective, highly sensitive technique offers potential for use in resource-limited clinical settings and can be easily adapted to differentiate between fungal species and detect drug resistance.

RevDate: 2024-10-28

Zhou Q, Gao Q, Gao Y, et al (2024)

BES-Designer: A Web Tool to Design Guide RNAs for Base Editing to Simplify Library.

Interdisciplinary sciences, computational life sciences [Epub ahead of print].

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.

RevDate: 2024-10-28

Hamlin JAP, Kozak-Muiznieks NA, Mercante JW, et al (2024)

Expanded geographic distribution for two Legionella pneumophila sequence types of clinical concern.

mSphere [Epub ahead of print].

Legionella pneumophila serogroup 1 sequence types (ST) 213 and 222, a single-locus variant of ST213, were first detected in the early 1990s in the Midwest United States (U.S.) and the late 1990s in the Northeast U.S. and Canada. Since 1992, these STs have increasingly been implicated in community-acquired sporadic and outbreak-associated Legionnaires' disease (LD) cases. We were interested in understanding the change in LD frequency due to these STs and identifying genetic features that differentiate these STs from one another. For the geographic area examined here (Mountain West to Northeast) and over the study period (1992-2020), ST213/222-associated LD cases identified by the Centers for Disease Control and Prevention increased by 0.15 cases per year, with ST213/222-associated LD cases concentrated in four states: Michigan (26%), New York (18%), Minnesota (16%), and Ohio (10%). Additionally, between 2002 and 2021, ST222 caused at least five LD outbreaks in the U.S.; no known outbreaks due to ST213 occurred in the U.S. during this time. We compared the genomes of 230 ST213/222 isolates and found that the mean of the average nucleotide identity (ANI) within each ST was high (99.92% for ST222 and 99.92% for ST213), with a minimum between ST ANI of 99.50% and a maximum of 99.87%, indicating low genetic diversity within and between these STs. While genomic features were identified (e.g., plasmids and CRISPR-Cas systems), no association explained the increasing geographic distribution and prevalence of ST213 and ST222. Yet, we provide evidence of the expanded geographical distribution of ST213 and ST222 in the U.S.IMPORTANCESince the 1990s, cases of Legionnaires' disease (LD) attributed to a pair of closely related Legionella pneumophila variants, ST213 and ST222, have increased in the U.S. Furthermore, between 2002 and 2021, ST222 caused at least five outbreaks of LD in the U.S., while ST213 has not been linked to any U.S. outbreak. We wanted to understand how the rate of LD cases attributed to these variants has changed over time and compare the genetic features of the two variants. Between 1992 and 2020, we determined an increase of 0.15 LD cases ascribed to ST213/222 per year in the geographic region studied. Our research shows that these STs are spreading within the U.S., yet most of the cases occurred in four states: Michigan, New York, Minnesota, and Ohio. Additionally, we found little genetic diversity within and between these STs nor could specific genetic features explain their geographic spread.

RevDate: 2024-10-29

Sharma N, Das A, Nair AV, et al (2024)

CRISPR-Cas system positively regulates virulence of Salmonella enterica serovar Typhimurium.

Gut pathogens, 16(1):63.

BACKGROUND: Salmonella, a foodborne pathogen, possesses a type I-E clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) system. We investigated the system's role in regulating Salmonella virulence by deleting the CRISPR arrays and Cas operon.

RESULTS: Our study demonstrates invasion and proliferation defects of CRISPR-Cas knockout strains in intestinal epithelial cells and macrophages owing to the repression of invasion and virulence genes. However, proliferation defects were not observed in the Gp91[phox-/-] macrophages, suggesting the system's role in the pathogens' antioxidant defense. We deduced that the CRISPR-Cas system positively regulates H2O2 importer (OmpW), catalase (katG), peroxidase (ahpC), and superoxide dismutase (soda and sodCI), thereby protecting the cells from oxidative radicals. The knockout strains were attenuated in in-vivo infection models (Caenorhabditis elegans and BALB/c mice) due to hypersensitivity against antimicrobial peptides, complement proteins, and oxidative stress. The attenuation in virulence was attributed to the suppression of LPS modifying (pmr) genes, antioxidant genes, master regulators, and effectors of the SPI-1 (invasion) and SPI-2 (proliferation) islands in knockout strains. The regulation could be attributed to the partial complementarity of the CRISPR spacers with these genes.

CONCLUSIONS: Overall, our study extends our understanding of the role of the CRISPR-Cas system in Salmonella pathogenesis and its virulence determinants.

RevDate: 2024-10-29
CmpDate: 2024-10-27

Ao Y, Gong X, Li J, et al (2024)

Characterization of NFDQ1 in Cryptosporidium parvum.

Parasites & vectors, 17(1):439.

BACKGROUND: Cryptosporidium spp. are important zoonotic parasites that can cause moderate to severe diarrhea in humans and animals. Among the three Cryptosporidium species infecting the intestines of calves, Cryptosporidium parvum has a broad host range and causes severe diarrhea in calves, while Cryptosporidium bovis and Cryptosporidium ryanae mainly infect calves without obvious clinical symptoms. Comparative genomic analysis revealed differences in the copy number of genes encoding the nonfinancial disclosure quality (NFDQ) secretory protein family among the three species, suggesting that this protein family may be associated with the host range or pathogenicity of Cryptosporidium spp. To understand the function of cgd8_10 encoded NFDQ1, tagged and knockout strains were constructed and characterized in this study.

METHODS: To determine the localization of NFDQ1, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to tag the C-terminus of NFDQ1 with three hemagglutinin epitopes (3 × HA). The tagged strain was constructed, and the genomic insertion was confirmed by polymerase chain reaction (PCR). Immunofluorescence assays were performed to observe the localization of NFDQ1 both in extracellular sporozoites and at various intracellular developmental stages. Immunoelectron microscopy was used to study the ultrastructural localization of NFDQ1. Then, the ΔNFDQ1 strain was generated by CRISPR/Cas9 and the in vitro growth assay on HCT-8 cells was used to analyze of phenotypic changes after knockout NFDQ1 in parasites.

RESULTS: The NFDQ1 tagging and knockout stains were successfully constructed by CRISPR/Cas9 technology and the insertions of transgenic strains were validated by PCR. The expression of NFDQ1 was validated in parasite by western blot. Immunofluorescence and immune-electron microscopy assay showed that NFDQ1 expressed in both asexual and sexual stages of C. parvum, where it was localized to the cytoplasm of the parasite. Upon ablation of NFDQ1, the ΔNFDQ1 strain showed an apparent growth retardation during sexual replication in vitro.

CONCLUSIONS: NFDQ1 is a cytoplasmic protein without specific localization to secretory organelles, and it may participate in C. parvum growth during sexual reproduction. Future study should determine the role of NFDQ1 following C. parvum infection in vivo.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Solano L (2024)

Novel gene therapies for sickle cell disease, Duchenne muscular dystrophy, and hemophilia A.

JAAPA : official journal of the American Academy of Physician Assistants, 37(11):17-22.

This article discusses novel genetic therapies for sickle cell disease, Duchenne muscular dystrophy, and hemophilia A. Gene therapies have the potential to deliver more targeted and effective approaches to treatment, especially for rare diseases for which the availability of approved therapies is limited. This article describes the first FDA-approved CRISPR/Cas9 treatment and the treatment protocols, indications, warnings, precautions, cost, and contraindications of four novel genetic therapies.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Meng S, Li Y, Dong N, et al (2024)

CRISPR/Cas12a-Sheared ZIF-Based Heterojunction to Allow Polarity-Switchable Photoelectrochemical and Nanozyme-Enabled Colorimetric Dual-Modal Biosensing.

Analytical chemistry, 96(43):17217-17226.

Modulating the migration of interfacial carriers in heterojunctions is critical for driving the signal response of high-performance optical biosensors. In this study, a polarity-switchable photoelectrochemical (PEC) and nanozyme-enabled colorimetric dual-modal biosensor is designed to modulate the interfacial carrier migration of the zeolitic imidazolate framework (ZIF)-based heterojunction by exploiting stem-loop DNA and the CRISPR/Cas12a system. Specifically, ZIF-hemin (ZIF-Hemin) is assembled at the CdSe/NH2-rGO interface via stem-loop DNA to form a ZIF-based heterojunction. Stem-loop DNA with a reinforcing rib effect enhances binding and accelerates the interfacial carrier migration of the heterojunction. In the presence of the target Cry1Ab, the CRISPR/Cas12a system is activated to shear the ZIF-based heterojunction, resulting in the disintegration of the heterojunction and the disappearance of interfacial carrier migration. At this point, ZIF-Hemin is released from the CdSe/NH2-rGO interface, with the photocurrent switching from the anode to the cathode. Meanwhile, due to its rich accessible active sites, the released ZIF-Hemin nanosheet shows high peroxidase-like catalytic activity and generates colorimetric signals. The dual-modal biosensor demonstrates excellent performance in selectivity and sensitivity, with low detection limits of 0.05 pg mL[-1] (PEC) and 0.4 pg mL[-1] (colorimetric). This work provides a general strategy to improve the performance of optical biosensors by modulating the migration of interfacial carriers in heterojunctions.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Campos Gudiño R, Neudorf NM, Andromidas D, et al (2024)

Loss of EMI1 compromises chromosome stability and is associated with cellular transformation in colonic epithelial cell contexts.

British journal of cancer, 131(9):1516-1528.

BACKGROUND: Colorectal cancer (CRC) is still a leading cause of cancer deaths worldwide. Thus, identifying the aberrant genes and proteins underlying disease pathogenesis is critical to improve early detection methods and develop novel therapeutic strategies. Chromosome instability (CIN), or ongoing changes in chromosome complements, is a predominant form of genome instability. It is a driver of genetic heterogeneity found in ~85% of CRCs. Although CIN contributes to CRC pathogenesis, the molecular determinants underlying CIN remain poorly understood. Recently, EMI1, an F-box protein, was identified as a candidate CIN gene. In this study, we sought to determine the impact reduced EMI1 expression has on CIN and cellular transformation.

METHODS: Coupling siRNA-based silencing and CRISPR/Cas9 knockout clones with quantitative imaging microscopy we evaluated the impact reduced EMI1 expression has on CIN and cellular transformation in four colonic epithelial cell contexts.

RESULTS: Quantitative imaging microscopy data revealed that reduced EMI1 expression induces increases in CIN phenotypes in both transient (siRNA) and constitutive (CRISPR/Cas9) cell models that are associated with increases in DNA damage and cellular transformation phenotypes in long-term studies.

CONCLUSIONS: This study determined that reduced EMI1 expression induces CIN and promotes cellular transformation, which is consistent with a role in early CRC development.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Zuo T, Shen C, Xie Z, et al (2024)

FRAME: flap endonuclease 1-engineered PAM module for precise and sensitive modulation of CRISPR/Cas12a trans-cleavage activity.

Nucleic acids research, 52(19):11884-11894.

CRISPR/Cas12a system, renowned for its precise recognition and efficient nucleic acid cleavage capabilities, has demonstrated remarkable performance in molecular diagnostics and biosensing. However, the reported Cas12a activity regulation methods often involved intricate CRISPR RNA (crRNA) structural adjustments or costly chemical modifications, which limited their applications. Here, we demonstrated a unique enzyme activity engineering strategy using flap endonuclease 1 (FEN1) to regulate the accessibility of the protospacer adjacent motif (PAM) module in the double-stranded DNA activator (FRAME). By identifying the three-base overlapping structure between the target inputs and substrate, FEN1 selectively cleaved and released the 5'-flap containing the 'TTTN' sequence, which triggered the secondary cleavage of FEN1 while forming a nicked PAM, ultimately achieving the sensitive switching of Cas12a's activity. The FRAME strategy exemplified the 'two birds with one stone' principle, as it not only precisely programmed Cas12a's activity but also simultaneously triggered isothermal cyclic amplification. Moreover, the FRAME strategy was applied to construct a sensing platform for detecting myeloperoxidase and miR-155, which demonstrated high sensitivity and specificity. Importantly, it proved its versatility in detecting multiple targets using a single crRNA without redesign. Collectively, the FRAME strategy opens up a novel avenue for modulating Cas12a's activity, promising immense potential in the realm of medical diagnostics.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Altinbay M, Wang J, Chen J, et al (2024)

Chem-CRISPR/dCas9FCPF: a platform for chemically induced epigenome editing.

Nucleic acids research, 52(19):11587-11601.

Epigenetic aberration is one of the major driving factors in human cancer, often leading to acquired resistance to chemotherapies. Various small molecule epigenetic modulators have been reported. Nonetheless, outcomes from animal models and clinical trials have underscored the substantial setbacks attributed to pronounced on- and off-target toxicities. To address these challenges, CRISPR/dCas9 technology is emerging as a potent tool for precise modulation of epigenetic mechanism. However, this technology involves co-expressing exogenous epigenetic modulator proteins, which presents technical challenges in preparation and delivery with potential undesirable side effects. Recently, our research demonstrated that Cas9 tagged with the Phe-Cys-Pro-Phe (FCPF)-peptide motif can be specifically targeted by perfluorobiphenyl (PFB) derivatives. Here, we integrated the FCPF-tag into dCas9 and established a chemically inducible platform for epigenome editing, called Chem-CRISPR/dCas9FCPF. We designed a series of chemical inhibitor-PFB conjugates targeting various epigenetic modulator proteins. Focusing on JQ1, a panBET inhibitor, we demonstrate that c-MYC-sgRNA-guided JQ1-PFB specifically inhibits BRD4 in close proximity to the c-MYC promoter/enhancer, thereby effectively repressing the intricate transcription networks orchestrated by c-MYC as compared with JQ1 alone. In conclusion, our Chem-CRISPR/dCas9FCPF platform significantly increased target specificity of chemical epigenetic inhibitors, offering a viable alternative to conventional fusion protein systems for epigenome editing.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Li HY, Wang M, Jiang X, et al (2024)

CRISPR screening uncovers nucleolar RPL22 as a heterochromatin destabilizer and senescence driver.

Nucleic acids research, 52(19):11481-11499.

Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Chen J, Su S, Pickar-Oliver A, et al (2024)

Engineered Cas9 variants bypass Keap1-mediated degradation in human cells and enhance epigenome editing efficiency.

Nucleic acids research, 52(19):11536-11551.

As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Singh M, Raseley K, Perez AM, et al (2024)

Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model.

Nucleic acids research, 52(19):11689-11703.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanism for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrate that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we show that targeting dCas9 to telomeres using sgTelo impedes DNA replication at telomeres and induces a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigate the genome-wide features of telomeres in the dCas9/sgTelo cells and observe a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-. Consistently, we also observe an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncover many interesting molecular and structural features of the ITCB and demonstrate that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Núñez-Álvarez Y, Espie-Caullet T, Buhagiar G, et al (2024)

A CRISPR-dCas13 RNA-editing tool to study alternative splicing.

Nucleic acids research, 52(19):11926-11939.

Alternative splicing allows multiple transcripts to be generated from the same gene to diversify the protein repertoire and gain new functions despite a limited coding genome. It can impact a wide spectrum of biological processes, including disease. However, its significance has long been underestimated due to limitations in dissecting the precise role of each splicing isoform in a physiological context. Furthermore, identifying key regulatory elements to correct deleterious splicing isoforms has proven equally challenging, increasing the difficulty of tackling the role of alternative splicing in cell biology. In this work, we take advantage of dCasRx, a catalytically inactive RNA targeting CRISPR-dCas13 ortholog, to efficiently switch alternative splicing patterns of endogenous transcripts without affecting overall gene expression levels cost-effectively. Additionally, we demonstrate a new application for the dCasRx splice-editing system to identify key regulatory RNA elements of specific splicing events. With this approach, we are expanding the RNA toolkit to better understand the regulatory mechanisms underlying alternative splicing and its physiological impact in various biological processes, including pathological conditions.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Meehan J, Ivens A, Grote S, et al (2024)

KREH2 helicase represses ND7 mRNA editing in procyclic-stage Trypanosoma brucei by opposite modulation of canonical and 'moonlighting' gRNA utilization creating a proposed mRNA structure.

Nucleic acids research, 52(19):11940-11959.

Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration. Canonical editing by gRNAs that specify protein-encoding mRNA sequences occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression at a major early checkpoint in mRNA ND7, involving helicase KREH2-dependent opposite modulation of canonical and non-canonical 'terminator' gRNA utilization. Terminator-programmed editing derails canonical editing and installs proposed repressive structure in 30% of the ND7 transcriptome. BSF-to-PCF differentiation in vitro recreated this negative control. Remarkably, KREH2-RNAi knockdown relieved repression and increased editing progression by reverting canonical/terminator gRNA utilization. ND7 transcripts lacking early terminator-directed editing in PCF exhibited similar negative editing control along the mRNA sequence, suggesting global modulation of gRNA utilization fidelity. The terminator is a 'moonlighting' gRNA also associated with mRNA COX3 canonical editing, so the gRNA transcriptome seems multifunctional. Thus, KREH2 is the first identified repressor in developmental editing control. This and our prior work support a model whereby KREH2 activates or represses editing in a stage and substrate-specific manner. KREH2's novel dual role tunes mitochondrial gene expression in either direction during development.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Xu C (2024)

CRISPR/Cas9-mediated knockout strategies for enhancing immunotherapy in breast cancer.

Naunyn-Schmiedeberg's archives of pharmacology, 397(11):8561-8601.

Breast cancer, a prevalent disease with significant mortality rates, often presents treatment challenges due to its complex genetic makeup. This review explores the potential of combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene knockout strategies with immunotherapeutic approaches to enhance breast cancer treatment. The CRISPR/Cas9 system, renowned for its precision in inducing genetic alterations, can target and eliminate specific cancer cells, thereby minimizing off-target effects. Concurrently, immunotherapy, which leverages the immune system's power to combat cancer, has shown promise in treating breast cancer. By integrating these two strategies, we can potentially augment the effectiveness of immunotherapies by knocking out genes that enable cancer cells to evade the immune system. However, safety considerations, such as off-target effects and immune responses, necessitate careful evaluation. Current research endeavors aim to optimize these strategies and ascertain the most effective methods to stimulate the immune response. This review provides novel insights into the integration of CRISPR/Cas9-mediated knockout strategies and immunotherapy, a promising avenue that could revolutionize breast cancer treatment as our understanding of the immune system's interplay with cancer deepens.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Bhushan B, Singh K, Kumar S, et al (2024)

Advancements in CRISPR-Based Therapies for Genetic Modulation in Neurodegenerative Disorders.

Current gene therapy, 25(1):34-45.

Neurodegenerative disorders pose significant challenges in the realm of healthcare, as these conditions manifest in complex, multifaceted ways, often attributed to genetic anomalies. With the emergence of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, a new frontier has been unveiled in the quest for targeted, precise genetic manipulation. This abstract explores the recent advancements and potential applications of CRISPR-based therapies in addressing genetic components contributing to various neurodegenerative disorders. The review delves into the foundational principles of CRISPR technology, highlighting its unparalleled ability to edit genetic sequences with unprecedented precision. In addition, it talks about the latest progress in using CRISPR to target specific genetic mutations linked to neurodegenerative diseases like Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Parkinson's disease. It talks about the most important studies and trials that show how well and safely CRISPR-based therapies work. This shows how this technology can change genetic variants that cause diseases. Notably, the discussion emphasizes the challenges and ethical considerations associated with the implementation of CRISPR in clinical settings, including off-target effects, delivery methods, and long-term implications. Furthermore, the article explores the prospects and potential hurdles in the widespread application of CRISPR technology for treating neurodegenerative disorders. It touches upon the need for continued research, improved delivery mechanisms, and ethical frameworks to ensure responsible and equitable access to these groundbreaking therapies.

RevDate: 2024-10-29
CmpDate: 2024-10-29

Khan A, Pudhuvai B, Shrestha A, et al (2024)

CRISPR-mediated iron and folate biofortification in crops: advances and perspectives.

Biotechnology & genetic engineering reviews, 40(4):4138-4168.

Micronutrient deficiency conditions, such as anemia, are the most prevalent global health problem due to inadequate iron and folate in dietary sources. Biofortification advancements can propel the rapid amelioration of nutritionally beneficial components in crops that are required to combat the adverse effects of micronutrient deficiencies on human health. To date, several strategies have been proposed to increase micronutrients in plants to improve food quality, but very few approaches have intrigued `clustered regularly interspaced short palindromic repeats' (CRISPR) modules for the enhancement of iron and folate concentration in the edible parts of plants. In this review, we discuss two important approaches to simultaneously enhance the bioavailability of iron and folate concentrations in rice endosperms by utilizing advanced CRISPR-Cas9-based technology. This includes the 'tuning of cis-elements' and 'enhancer re-shuffling' in the regulatory components of genes that play a vital role in iron and folate biosynthesis/transportation pathways. In particular, base-editing and enhancer re-installation in native promoters of selected genes can lead to enhanced accumulation of iron and folate levels in the rice endosperm. The re-distribution of micronutrients in specific plant organs can be made possible using the above-mentioned contemporary approaches. Overall, the present review discusses the possible approaches for synchronized iron and folate biofortification through modification in regulatory gene circuits employing CRISPR-Cas9 technology.

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ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
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Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin and even a collection of poetry — Chicago Poems by Carl Sandburg.

Timelines

ESP now offers a large collection of user-selected side-by-side timelines (e.g., all science vs. all other categories, or arts and culture vs. world history), designed to provide a comparative context for appreciating world events.

Biographies

Biographical information about many key scientists (e.g., Walter Sutton).

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 28 JUL 2024 )