CRISPR-associated Protein Research Articles

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.

Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.

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

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.

RNA processing by the CRISPR-associated NYN ribonuclease.

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

ACURAT: Advancing tuberculosis detection through assembled PCR & CRISPR for ultra-sensitive Rifampin-resistant analysis testing

Tuberculosis (TB) remains a pressing global health threat, while the emergence of drug-resistant TB adds complexity to clinical treatment strategies, underscoring the need for rapid and accurate diagnosis. In this study, we present an innovative identification and antibiotic susceptibility testing system, termed Assembled PCR & CRISPR for Ultra-sensitive Rifampin-resistant Analysis Testing (ACURAT). ACURAT employed a nested polymerase chain reaction (PCR) scheme to sensitively detect as few as 10 copies of the TB genome, more importantly, addressing the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR-Cas9) system’s requirement for a protospacer adjacent motif (PAM) sequence by introducing with the designed primers. Recognizing the limitation of fully-matched guide ribonucleic acid (RNA) in distinguishing one-base single nucleotide polymorphisms (SNPs), we designed a micro-mismatch CRISPR-Cas9 system with significant selectivity, featuring a single-base mismatch with wildtype genotypes and at least two base mismatches with drug-resistant genotypes. The systematic optimization of ACURAT parameters revealed that a 19 nt guide RNA length, a 1 bp distance to the PAM sequence, and the use of a high-fidelity Cas9 nuclease significantly enhanced the efficiency. Finally, ACURAT successfully distinguished fifteen drug-resistance-related genotypes in codons 516, 526, 531, and 533 of the polymerase beta subunit gene. ACURAT demonstrated substantial clinical potential for efficient TB diagnosis and exhibited the capability to address other sequence recognition scenarios.

Clustered Regularly Interspaced Short Palindromic Repeat-1 (CRISPR-1) Locus as a Tool for Tracing the Zoonotic History of Salmonella enterica Strains.

Background Salmonella enterica is a significant foodborne pathogen that causes considerable illness and death in humans and animals. The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system in bacteria acts as an adaptive immune defense against invasive genetic elements by incorporating short intergenic spacers (IGSs) into CRISPR loci. These loci serve as molecular records of past interactions with phages and plasmids, providing insights into the transmission and evolution of bacterial strains across different hosts. Aim This study aimed to investigate the diversity of IGSs in the CRISPR-1 locus of S. enterica isolates from humans and camels. The objective was to assess the potential of IGSs to distinguish strains, track sources, and understand patterns of zoonotic transmission. Materials and methods Genomic DNA was extracted from multiple strains of S. enterica, and the CRISPR-1 locus was polymerase chain reaction (PCR) amplified and sequenced. The sequences were compared to identify distinct patterns of IGSs and potential host-specific characteristics. Sanger sequencing and bioinformatics tools were used to classify the IGSs and determine their similarity to known sequences in the National Center for Biotechnology Information (NCBI) database. Results Sequence analysis revealed five distinct CRISPR-1 types among S. enterica isolates from humans and three among camel isolates. The presence of shared IGSs between human and camel S. entericaisolates suggested zoonotic or reverse-zoonotic transmission events. Additionally, host-specific unknown IGSs (UIGS) were identified. Importantly, camel isolates initially identified as S. enterica subspeciesenterica serovar Enteritidis based on rrnH gene sequencing were reclassified as S. enterica serovar Enteritidis based on CRISPR-1 profiling, demonstrating the higher resolution of CRISPR-based genotyping. Conclusion The diversity of IGSs in the CRISPR-1 locus effectively differentiated S. enterica strains and provided insights into their evolutionary origins and transmission dynamics. CRISPR-based genotyping proves to be a promising tool to complement traditional serotyping methods, enhancing the molecular epidemiology of salmonellosis and potentially leading to better management and control strategies for this pathogen.

Discovery and engineering of ChCas12b for precise genome editing

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.

Neuropeptide Y receptor Y8b (npy8br) regulates feeding and digestion in Japanese medaka (Oryzias latipes) larvae: evidence from gene knockout.

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br-/- Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br-/- medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br-/- medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

Comprehensive Analysis of Antiphage Defense Mechanisms: Serovar-Specific Patterns.

Leptospirosis is a major zoonotic disease caused by pathogenic spirochetes in the genus Leptospira, affecting over a million people annually and causing approximately 60,000 deaths. Leptospira interrogans, a key causative agent, likely possesses defense systems against bacteriophages (leptophages), yet these systems are not well understood. We analyzed 402 genomes of L. interrogans using the DefenseFinder tool to identify and characterize the antiphage defense systems. We detected 24 unique systems, with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins), PrrC, Borvo, and Restriction-Modification (R-M) being the most prevalent. Notably, Cas were identified in all strains, indicating their central role in phage defense. Furthermore, there were variations in the antiphage system distribution across different serovars, suggesting unique evolutionary adaptations. For instance, Retron was found exclusively in the Canicola serovar, while prokaryotic Argonaute proteins (pAgo) were only detected in the Grippotyphosa serovar. These findings significantly enhance our understanding of Leptospira's antiphage defense mechanisms. They reveal the potential for the development of serovar-specific phage-based therapies and underscore the importance of further exploring these defense systems.

CRISPR/Cas9 Technology: A Novel Approach to Obesity Research.

Gene editing technology, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has transformed medical research. As a newly developed genome editing technique, CRISPR technology has strongly assisted scientists in enriching their comprehension of the roles of individual genes and their influences on a vast spectrum of human malignancies. Despite considerable progress in elucidating obesity's molecular pathways, current anti-obesity medications fall short in effectiveness. A thorough understanding of the genetic foundations underlying various neurobiological pathways related to obesity, as well as the neuro-molecular mechanisms involved, is crucial for developing effective obesity treatments. Utilizing CRISPR-based technologies enables precise determination of the roles of genes that encode transcription factors or enzymes involved in processes, such as lipogenesis, lipolysis, glucose metabolism, and lipid storage within adipose tissue. This innovative approach allows for the targeted suppression or activation of genes regulating obesity, potentially leading to effective weight management strategies. In this review, we have provided a detailed overview of obesity's molecular genetics, the fundamentals of CRISPR/Cas9 technology, and how this technology contributes to the discovery and therapeutic targeting of new genes associated with obesity.

High-resolution genome-wide mapping of chromosome-arm-scale truncations induced by CRISPR–Cas9 editing

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) is a powerful tool for introducing targeted mutations in DNA, but recent studies have shown that it can have unintended effects such as structural changes. However, these studies have not yet looked genome wide or across data types. Here we performed a phenotypic CRISPR–Cas9 scan targeting 17,065 genes in primary human cells, revealing a ‘proximity bias’ in which CRISPR knockouts show unexpected similarities to unrelated genes on the same chromosome arm. This bias was found to be consistent across cell types, laboratories, Cas9 delivery methods and assay modalities, and the data suggest that it is caused by telomeric truncations of chromosome arms, with cell cycle and apoptotic pathways playing a mediating role. Additionally, a simple correction is demonstrated to mitigate this pervasive bias while preserving biological relationships. This previously uncharacterized effect has implications for functional genomic studies using CRISPR–Cas9, with applications in discovery biology, drug-target identification, cell therapies and genetic therapeutics.

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling.

Hepatic fibrosis (HF) is a critical step in the progression of hepatocellular carcinoma (HCC). Gene associated with retinoid-IFN-induced mortality 19 (GRIM19), an essential component of mitochondrial respiratory chain complex I, is frequently attenuated in various human cancers, including HCC. Here, we aimed to investigate the potential relationship and underlying mechanism between GRIM19 loss and HF pathogenesis. GRIM19 expression was evaluated in normal liver tissues, hepatitis, hepatic cirrhosis, and HCC using human liver disease spectrum tissue microarrays. We studied hepatocyte-specific GRIM19 knockout mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) lentivirus-mediated GRIM19 gene-editing in murine hepatocyte AML12 cells in vitro and in vivo. We performed flow cytometry, immunofluorescence, immunohistochemistry, western blotting, and pharmacological intervention to uncover the potential mechanisms underlying GRIM19 loss-induced HF. Mitochondrial GRIM19 was progressively downregulated in chronic liver disease tissues, including hepatitis, cirrhosis, and HCC tissues. Hepatocyte-specific GRIM19 heterozygous deletion induced spontaneous hepatitis and subsequent liver fibrogenesis in mice. In addition, GRIM19 loss caused chronic liver injury through reactive oxygen species (ROS)-mediated oxidative stress, resulting in aberrant NF-кB activation via an IKK/IкB partner in hepatocytes. Furthermore, GRIM19 loss activated NLRP3-mediated IL33 signaling via the ROS/NF-кB pathway in hepatocytes. Intraperitoneal administration of the NLRP3 inhibitor MCC950 dramatically alleviated GRIM19 loss-driven HF in vivo. The mitochondrial GRIM19 loss facilitates liver fibrosis through NLRP3/IL33 activation via ROS/NF-кB signaling, providing potential therapeutic approaches for earlier HF prevention.

Label-free Colorimetric Detection of Viral RNA Based on Clustered Regularly Interspaced Short Palindromic Repeats and Gold Nanoparticles with a Portable Device.

Viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are some of the most dangerous threats to humans. SARS-CoV-2 has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. To meet these requirements, we designed a label-free colorimetric platform that combines the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) 12a system for naked-eye detection (named LFP). This method utilizes reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the trans-cleavage activity of the CRISPR/Cas12a system to increase the sensitivity and specificity of the reaction. This platform can detect as few as 4 copies/μL of RNA and produces no false positive results when tested against the influenza virus. To better meet the requirements of point-of-care (POC) detection, we developed a portable device that can be applied in resource-poor and densely populated regions. The LFP assay holds great potential for application in resource-limited settings, and the label-free gold nanoparticle (AuNPs) probe can reduce costs, making it suitable for large-scale screening. We expect that the LFP assay will be promising for the POC screening of COVID-19.

Delivering CRISPR to the HIV-1 reservoirs.

Human immunodeficiency virus type 1 (HIV-1) infection is well known as one of the most complex and difficult viral infections to cure. The difficulty in developing curative strategies arises in large part from the development of latent viral reservoirs (LVRs) within anatomical and cellular compartments of a host. The clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system shows remarkable potential for the inactivation and/or elimination of integrated proviral DNA within host cells, however, delivery of the CRISPR/Cas9 system to infected cells is still a challenge. In this review, the main factors impacting delivery, the challenges for delivery to each of the LVRs, and the current successes for delivery to each reservoir will be discussed.

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments.

In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome.

FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.

Gene Editing for CEP290-Associated Retinal Degeneration

BackgroundCEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant).MethodsWe performed a phase 1–2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora–Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire–25 (in adults) or the Children’s Visual Function Questionnaire (in children).ResultsEDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score.ConclusionsThe safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1–2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.)

Flavomycin restores colistin susceptibility in multidrug-resistant Gram-negative bacteria.

Colistin is a critical antibiotic in combating multi-drug resistant Gram-negative bacteria, but the emergence of mobilized colistin resistance (mcr) undermines its effectiveness. Previous studies have found that colistin can synergy with various drugs; however, its exact mechanisms with hydrophobic drugs are still unrevealed. Generally, the membrane destruction of colistin is thought to be the essential trigger for its interactions with its partner drugs. Here, we use clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) for specifically mutating the binding site of one hydrophobic drug (flavomycin) and show that antimicrobial activity of flavomycin is critical for the synergy. Our results first give the evidence that the synergy is set off by colistin's membrane destruction and operated the final antimicrobial function by its partner drugs.

Effects of plumbagin on proliferation and apoptosis of human hepatocellular carcinoma cells based on CKB/p53 signaling pathway

To investigate the effects of plumbagin on the proliferation and apoptosis of human hepatoma Huh-7 cells and its mechanism based on the creatine kinase B(CKB)/p53 signaling pathway. Huh-7 cells were treated with plumbagin from 1 to 12 μmol·L~(-1) for cell counting kit-8(CCK-8) assay, and 1, 3, and 6 μmol·L~(-1) were determined as low, medium, and high concentrations of plumbagin for subsequent experiments. CKB gene was knocked out in Huh-7 cells by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated proteins(Cas)-9 gene editing technology. CKB overexpression lentivirus was transfected into Huh-7 cells to up-regulate the expression of CKB. Cell proliferation and apoptosis were detected by plate cloning assay and flow cytometry. The mRNA expression of CKB was detected by quantitative real-time PCR(qRT-PCR). CKB, p53, mouse double minute 2 homolog(MDM2), B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), and caspase-3 protein were detected by Western blot(WB). The results showed that plumbagin significantly inhibited the proliferation of Huh-7 cells and induced cell apoptosis. Compared with the control group, the apoptosis level was significantly increased in the plumbagin group, while the apoptosis level was significantly decreased in the plumbagin combined with the apoptosis inhibitor group. Plumbagin significantly down-regulated the protein expression levels of CKB, Bcl-2, and MDM2 and up-regulated the protein expression levels of p53, Bax, and caspase-3. Knockdown of the CKB gene decreased the proliferative ability of Huh-7 cells, increased the apoptotic rate, decreased the expression levels of Bcl-2 and MDM2 proteins, and increased the expression levels of p53, Bax, and caspase-3 proteins. After up-regulation of CKB expression, the proliferation ability of Huh-7 cells was enhanced, and the protein expression levels of Bcl-2 and MDM2 were elevated. The protein expression levels of p53, Bax, and caspase-3 were decreased. In addition, plumbagin reversed the effect of overexpression of CKB on the proliferation and apoptosis of Huh-7 cells. In conclusion, plumbagin significantly inhibited the proliferative ability of Huh-7 cells, and the mechanism may be related to the inhibition of CKB expression, activation of the p53 signaling pathway, and regulation of the expression of mitochondrial-associated apoptotic proteins, ultimately inducing cell apoptosis.

Reproductive Characteristics and Suitability of Sterile dead end Knockout Nibe Croaker as a Recipient for Intraperitoneal Germ Cell Transplantation.

The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.