CRISPR Research Articles

Genome Editing of Mammalian Cells Through RNA Transcript-Mediated Homologous Recombination Repair.

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

Blue valorization of lignin-derived monomers via reprogramming marine bacterium Roseovarius nubinhibens.

Biological valorization of lignin, the second most abundant biopolymer on Earth, is an indispensable sector to build a circular economy and net-zero future. However, lignin is recalcitrant to bioupcycling, demanding innovative solutions. We report here the biological valorization of lignin-derived aromatic carbon to value-added chemicals without requesting extra organic carbon and freshwater via reprogramming the marine Roseobacter clade bacterium Roseovarius nubinhibens. We discovered the unusual advantages of this strain for the oxidation of lignin monomers and implemented a CRISPR interference (CRISPRi) system with the lacI-Ptrc inducible module, nuclease-deactivated Cas9, and programmable gRNAs. This is the first CRISPR-based regulatory system in R. nubinhibens, enabling precise and efficient repression of genes of interest. By deploying the customized CRISPRi, we reprogrammed the carbon flux from a lignin monomer, 4-hydroxybenzoate, to achieve the maximum production of protocatechuate, a pharmaceutical compound with antibacterial, antioxidant, and anticancer properties, with minimal carbon to maintain cell growth and drive biocatalysis. As a result, we achieved a 4.89-fold increase in protocatechuate yield with a dual-targeting CRISPRi system, and the system was demonstrated with real seawater. Our work underscores the power of CRISPRi in exploiting novel microbial chassis and will accelerate the development of marine synthetic biology. Meanwhile, the introduction of a new-to-the-field lineage of marine bacteria unveils the potential of blue biotechnology leveraging resources from the ocean.IMPORTANCEOne often overlooked sector in carbon-conservative biotechnology is the water resource that sustains these enabling technologies. Similar to the "food-versus-fuel" debate, the competition of freshwater between human demands and bioproduction is another controversial issue, especially under global water scarcity. Here, we bring a new-to-the-field lineage of marine bacteria with unusual advantages to the stage of engineering biology for simultaneous carbon and water conservation. We report the valorization of lignin monomers to pharmaceutical compounds without requesting extra organic substrate (e.g., glucose) or freshwater by reprogramming the marine bacterium Roseovarius nubinhibens with a multiplex CRISPR interference system. Beyond the blue lignin valorization, we present a proof-of-principle of leveraging marine bacteria and engineering biology for a sustainable future.

Next-Generation strategies to combat antimicrobial resistance: Integrating genomics, CRISPR, and novel therapeutics for effective treatment

Antimicrobial resistance (AMR) poses a significant threat to global public health, necessitating innovative strategies to combat this escalating issue. This review outlines next-generation approaches integrating genomics, CRISPR technology, and novel therapeutics to effectively address AMR. Genomic techniques enable comprehensive understanding of the genetic mechanisms underpinning resistance, facilitating the development of targeted interventions. By sequencing the genomes of resistant pathogens, researchers can identify resistance genes, track their spread, and predict emerging resistance patterns. CRISPR-Cas systems offer a revolutionary tool for combating AMR through precise genome editing. This technology can disrupt resistance genes, restore antibiotic sensitivity, and develop bacteriophage therapies that selectively target resistant bacteria. Moreover, CRISPR-based diagnostics enable rapid, accurate detection of resistant strains, enhancing infection control measures. The advent of novel therapeutics, such as antimicrobial peptides, bacteriophage therapy, and synthetic biology-derived compounds, provides alternative treatment options. These therapeutics can bypass traditional resistance mechanisms and exhibit efficacy against multi-drug resistant organisms. Additionally, integrating artificial intelligence (AI) and machine learning with genomics and CRISPR can accelerate the discovery of new antibiotics and predict resistance trends, optimizing treatment regimens. Implementing these next-generation strategies requires robust global collaboration, regulatory frameworks, and investment in research and development. By combining genomics, CRISPR, and novel therapeutics, we can create a multifaceted approach to overcome AMR, ensuring effective treatments and safeguarding public health. This integration represents a paradigm shift in antimicrobial strategy, offering hope for a future where resistant infections can be effectively managed and treated. Keywords: Integrating Genomics, Antimicrobial Resistance, CRISPR, Therapeutic

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

Progress of CRISPR Technology in Cancer Treatment

Progress of CRISPR Technology in Cancer Treatment

Unlocking Rapid and User-Friendly Strategies to Improve Horticultural Crop Qualities

Climatic changes and global warming affect the growth, development, and productivity of crops. In this review, we highlight the possible benefits of using innovative breeding techniques like clustered regularly interspaced short palindromic repeats (CRISPRs), exogenous phytohormone-like strigolactones (SLs), nanomaterials (NMs), and beneficial microbial endophytes to address the challenges in sustainable cultivation of horticultural crops. These applications are evaluated by examining how they affect different metabolic, morphological, and biochemical parameters in diverse crops. Endophytes are symbiotic microorganisms and can be used as nematicides for improving crop yield. With an emphasis on quality control, we examined the impacts of applying NMs, a novel family of phytohormones called SLs, and microbial endophytes on horticultural commodities. Furthermore, we reviewed the benefits of CRISPR for the editing of plant genomes, as well as how it affects gene expression and transcription factors to increase crop tolerance and yield. These innovations hold the potential to improve crop yield, quality, and resilience by acting as safe, natural components in biofertilizers and plant protection solutions. Gradually adopting these methods could decrease reliance on agrochemicals, thereby reducing their negative effects on biodiversity, soil fertility, and human health.

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

CRISPR-based genetic screens advance cancer immunology.

CRISPR technologies have revolutionized research areas ranging from fundamental science to translational medicine. CRISPR-based genetic screens offer a powerful platform for unbiased screening in various fields, such as cancer immunology. Immune checkpoint blockade (ICB) therapy has been shown to strongly affect cancer treatment. However, the currently available ICBs are limited and do not work in all cancer patients. Pooled CRISPR screens enable the identification of previously unknown immune regulators that can regulate T-cell activation, cytotoxicity, persistence, infiltration into tumors, cytokine secretion, memory formation, T-cell metabolism, and CD4+ T-cell differentiation. These novel targets can be developed as new immunotherapies or used with the current ICBs as new combination therapies that may yield synergistic efficacy. Here, we review the progress made in the development of CRISPR technologies, particularly technological advances in CRISPR screens and their application in novel target identification for immunotherapy.

Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Genetic Engineering and Innovative Cultivation Strategies for Enhancing the Lutein Production in Microalgae.

Carotenoids, with their diverse biological activities and potential pharmaceutical applications, have garnered significant attention as essential nutraceuticals. Microalgae, as natural producers of these bioactive compounds, offer a promising avenue for sustainable and cost-effective carotenoid production. Despite the ability to cultivate microalgae for its high-value carotenoids with health benefits, only astaxanthin and β-carotene are produced on a commercial scale by Haematococcus pluvialis and Dunaliella salina, respectively. This review explores recent advancements in genetic engineering and cultivation strategies to enhance the production of lutein by microalgae. Techniques such as random mutagenesis, genetic engineering, including CRISPR technology and multi-omics approaches, are discussed in detail for their impact on improving lutein production. Innovative cultivation strategies are compared, highlighting their advantages and challenges. The paper concludes by identifying future research directions, challenges, and proposing strategies for the continued advancement of cost-effective and genetically engineered microalgal carotenoids for pharmaceutical applications.

CRISPR-Cas System, Antimicrobial Resistance, and Enterococcus Genus-A Complicated Relationship.

(1) Background: The rise in antibiotic resistant bacteria poses a significant threat to public health worldwide, necessitating innovative solutions. This study explores the role of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the context of antibiotic resistance among different species from the Enterococcus genus. (2) Methods: The genomes of Enterococcus included in the study were analyzed using CRISPRCasFinder to distinguish between CRISPR-positive (level 4 CRISPR) and CRISPR-negative genomes. Antibiotic resistance genes were identified, and a comparative analysis explored potential associations between CRISPR presence and antibiotic resistance profiles in Enterococcus species. (3) Results: Out of ten antibiotic resistance genes found in Enterococcus species, only one, the efmA gene, showed a strong association with CRISPR-negative isolates, while the others did not significantly differ between CRISPR-positive and CRISPR-negative Enterococcus genomes. (4) Conclusion: These findings indicate that the efmA gene may be more prevalent in CRISPR-negative Enterococcus genomes, and they may contribute to a better understanding of the molecular mechanisms underlying the acquisition of antibiotic resistance genes in Enterococcus species.

An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.

Targeting Tumor-Associated Sialic Acids Using Chimeric Switch Receptors Based on Siglec-9 Enhances the Antitumor Efficacy of Engineered T Cells.

Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment. See related Spotlight by Abken, p. 1310.

EfpA is required for regrowth of Mycobacterium tuberculosis following isoniazid exposure.

Efflux of antibiotics is an important survival strategy in bacteria. Mycobacterium tuberculosis has approximately sixty efflux pumps, but little is known about the role of each pump or the substrates they efflux. The putative efflux pump, EfpA, is a member of the major facilitator superfamily and has been shown to be essential by saturation transposon mutagenesis studies. It has been implicated in the efflux of isoniazid (INH), which is a first-line drug used to treat tuberculosis (TB). This is supported by evidence from transcriptional profiling showing that efpA is induced in response to INH exposure. However, its roles in the physiology and adaptation of M. tuberculosis to antibiotics have yet to be determined. In this study, we describe the repression of efpA in M. tuberculosis, using CRISPR interference (CRISPRi) to knockdown the expression of this essential gene and the direct effect of this on the ability of M. tuberculosis to survive exposure to INH over a 45-day time course. We determined that wild-type levels of efpA were required for recovery of M. tuberculosis following INH exposure and that, after 45 days of INH exposure, only a few viable colonies were recoverable from efpA-repressed M. tuberculosis. We conclude that EfpA is required for recovery of M. tuberculosis following INH exposure, which could reduce the efficacy of INH in vivo, and that EfpA may have a role in the development of resistance during drug therapy.

Peptide Nucleic Acid-Mediated Regulation of CRISPR-Cas9 Specificity.

Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR's application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.

Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the SARS-CoV-2 target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. Comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structure than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

Epidemiological and molecular investigations of Salmonella isolated from duck farms in southwest and around area of Shandong, China

Salmonella is a zoonotic pathogen posing a serious risk to the farming industry and public health due to food animals serving as reservoirs for future contamination and spread of Salmonella. The present study is designed to monitor the contamination status of Salmonella in duck farms and the main control points during breeding. 160 strains of duck-derived Salmonella were isolated from the 736 samples (cloacal swabs, feces, water, feed, soil, air and dead duck embryos) collected in southwest Shandong Province and the province's surrounding area. The percentage of Salmonella-positive samples collected was 21.74 % (160/736), and the greatest prevalence from duck embryo samples (40.00 %, 36/90). These Salmonella were classified into 23 serotypes depending on their O and H antigens, in which S. Typhimurium (30.15 %), S. Kottbus (13.97 %) and S. Enteritidis (10.29 %) were the prevailing serotypes. Subsequently, the molecular subtyping was done. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis showed that 41 strains of S. Typhimurium and 14 strains of S. Enteritidis were classified into 13 and 3 genotypes, respectively. 19 S. Kottbus isolates from different sources featured ST1546, ST198, ST321, and ST1690 by multilocus sequence typing (MLST) analysis, among which ST1546 belongs to S. Kottbus was a new ST. The minimum spanning tree analysis based on the two CRISPR loci and seven MLST loci from all S. Typhimurium, S. Enteritidis and S. Kottbus isolates revealed that duck embryos, feed and water were key control points to the spread of Salmonella along the breeding chain. Meanwhile, the emergence of S. Kottbus in duck flocks was considered a potential public health hazard.

Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson's disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.

A Review: CRISPR Cas System and the Mechanism With an Inhibition of Binding of CRISPR Cas‐9

A Review: CRISPR Cas System and the Mechanism With an Inhibition of Binding of CRISPR Cas‐9

CRISPR-Cas12a exhibits metal-dependent specificity switching.

Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.