Regularly-interspaced Short Palindromic Repeats Research Articles

Clearance of residual genome editing components used for ex vivo genome-editing of allogeneic cell therapy products

Background aimsClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–associated genome editing (GE) components (e.g., nucleases, guide RNAs (gRNAs), and plasmids) are used to genetically modify cells during development of ex vivo genome-edited cell therapies. Prolonged presence of GE components may increase the risk of unintended genome modifications (e.g., off-target editing and chromosomal rearrangements). This risk is a function of the stability of the GE components, culture conditions (i.e., culture length, media changes, etc.), and the nature of the GE component (i.e., only plasmids can be integrated into a cell's genome). Testing for residual GE components on ex vivo genetically edited drug products is generally recommended in current regulatory guidance (CBER 2024).For allogenic cell therapies derived from induced pluripotent stem cells (iPSC), cells typically undergo clonal selection and extensive culturing following completion of genome editing. This post-engineering clonal selection substantially reduces the amount of residual GE components while the long-duration cell culture significantly reduces the presence of active residual GE components. Here we present a case in which the need for testing of the drug product for residual GE components has been eliminated. MethodsIn silico modeling was used to estimate clearance mechanisms across a variety of relevant assumptions, including disposition of extracellular GE components via media changes and dilution of intracellular GE components via cell expansion. Determining the ability of each GE component—alone or in complex with other GE components—to modify genomic material was assessed by a series of both in vitro and ex vivo (i.e., engineering cells) studies. For the in vitro studies, a DNA cutting assay was developed to assess the ability of the component to cut a representative DNA strand. For the ex vivo modification of cells, an assessment of the knock-out of the relevant gene was completed by flow cytometry specifically assessing the presence or absence of protein expression on the modified cells. The persistence and stability of GE components were examined under cell-mimicking conditions and in ex vivo modified cells. The components were stressed under multiple conditions mimicking a range of culture conditions and tested in the aforementioned DNA cutting assay. The presence of residual gRNA was directly assessed in the ex vivo modified cells via a gRNA-specific digital droplet polymerase chain reaction (ddPCR) assay. ResultsSimulations estimating genome editing residual clearance via dilution for extracellular residuals (via media changes) or intracellular residuals (via cell doubling) demonstrate clearance of measurable residuals within 28 days of cell culture. Studies simulating the stability of genome editing residuals estimate less than 7 days for the nuclease, gRNA and ribonucleoprotein (RNP) complex. gRNA was undetectable by 8 days post-engineering under actual engineering conditions. Additionally, without gRNA present, CRISPR Cas12a nucleases did not demonstrate evidence of cutting.In contrast, plasmid DNA can be randomly integrated into the genome and free plasmid is highly stable under cell culture-like conditions (50+ days). Additionally, plasmid DNA integrated in cells will propagate during mitosis, leading to the additional risk of expansion of an unintentional integration event. ConclusionsBoth the gRNA and nuclease in the RNP complex are required for DNA cutting. Neither individual component nor the complex are stable beyond 7 days in culture-mimicking conditions. These findings suggest that the risk of unplanned genomic modification resulting from residual gRNA or nuclease is minimal for processes in which extensive culture is performed after the completion of genome editing and clonal selection. However, the risk of residual plasmid DNA integration is significantly higher regardless of the manufacturing process. The residual plasmid itself is quite stable (at least 50 days) and the risk of random, off-target integration is present. By establishing the stability of these components, we have demonstrated that testing for residual gRNA or nuclease is not warranted for clonally derived allogeneic cell therapies.

Toward the Development of a Tissue Engineered Gradient Utilizing CRISPR-Guided Gene Modulation.

Cellular, compositional, and mechanical gradients are found throughout biological tissues, especially in transition zones between tissue types. Yet, strategies to engineer such gradients have proven difficult due to the complex nature of these tissues. Current strategies for tissue engineering complex gradients often utilize stem cells; however, these multipotent cells require direction from environmental cues, which can be difficult to control both in vitro and in vivo. In this study, we utilize clustered regularly-interspaced short palindromic repeats (CRISPR)-guided gene modulation to direct the differentiation of multipotent adipose-derived stem cells (ASCs) to demonstrate the effectiveness of CRISPR-engineered cells in tissue engineering applications. Specifically, we screen CRISPR-interference (CRISPRi) constructs targeting the promotors of selected osteogenic inhibitors and demonstrate that ASC osteogenic differentiation and mineral deposition can be regulated with CRISPRi targeting of Noggin without the use of exogenous growth factors in tissue engineered constructs. As a proof of concept, we combine three technologies developed out of our laboratories to demonstrate the controlled deposition of these engineered cells in a gradient with CRISPR-activation multiplex-engineered aggrecan/collagen type-II-chondrogenic ASCs on a high density anisotropic type I collagen construct to create a cell and tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the enthesis. Our results display the promise of CRISPR-engineered ASCs to produce tissue gradients, similar to what is observed in native tissue.

Applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a Genetic Scalpel for the Treatment of Cancer: A Translational Narrative Review.

Cancer remains a global health challenge with high prevalence and mortality rates, imposing significant financial and emotional burdens on affected families. However, hope lies in genetic manipulation, with a focus on innovative approaches to combat genetically linked cancers. Clustered regularly interspaced short palindromic repeats (CRISPR), an adaptive immune system found in various bacteria and archaea, hold immense potential. We searched articles on PubMed Central, Medline, and PubMed databasesindexed journals. The keywords from the research topic, i.e., "CRISPR AND genetic therapy," were searched, and we found 3397 articles. Following this, we explored the medical subject headings (MeSH) glossary and created a search strategy "Clustered Regularly Interspaced Short Palindromic Repeats"[Mesh] AND "Genetic Therapy"[Majr] and after applying a variety of filters we included 30 studies in our review. CRISPR consists of unique spacers and CRISPR-associated (Cas) genes, operating through acquisition, CRISPR ribonucleic acid (crRNA) biogenesis, and target interference phases. The type II CRISPR-Cas9 system is a well-researched avenue for gene editing, with Cas9 cleaving specific genomic regions and initiating deoxyribonucleic acid (DNA) repair mechanisms. Cancer results from genetic alterations, leading to tumor development with properties like metastasis. CRISPR/Cas9 offers precise genome editing to inhibit tumor formation by removing specific genomic sequences and promoting DNA repair. Challenges in CRISPR's use for cancer therapy, including delivery methods, cell adaptation, and ethical concerns, are recognized. Beyond cancer, CRISPR finds diverse applications in infectious diseases and non-cancerous conditions, signifying its transformative potential in modern medicine. CRISPR technology represents a groundbreaking frontier in cancer therapy and beyond, offering insights into genetic editing, disease mechanisms, and potential cures. Despite challenges and ethical considerations, precise genome editing promises improved cancer treatments and innovative medical interventions in the future.

Herbgenomics meets Papaveraceae: a promising -omics perspective on medicinal plant research.

Herbal medicines were widely used in ancient and modern societies as remedies for human ailments. Notably, the Papaveraceae family includes well-known species, such as Papaver somniferum and Chelidonium majus, which possess medicinal properties due to their latex content. Latex-bearing plants are a rich source of diverse bioactive compounds, with applications ranging from narcotics to analgesics and relaxants. With the advent of high-throughput technologies and advancements in sequencing tools, an opportunity exists to bridge the knowledge gap between the genetic information of herbs and the regulatory networks underlying their medicinal activities. This emerging discipline, known as herbgenomics, combines genomic information with other -omics studies to unravel the genetic foundations, including essential gene functions and secondary metabolite biosynthesis pathways. Furthermore, exploring the genomes of various medicinal plants enables the utilization of modern genetic manipulation techniques, such as Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR/Cas9) or RNA interference. This technological revolution has facilitated systematic studies of model herbs, targeted breeding of medicinal plants, the establishment of gene banks and the adoption of synthetic biology approaches. In this article, we provide a comprehensive overview of the recent advances in genomic, transcriptomic, proteomic and metabolomic research on species within the Papaveraceae family. Additionally, it briefly explores the potential applications and key opportunities offered by the -omics perspective in the pharmaceutical industry and the agrobiotechnology field.

Removing Harmful Pericarp Character of Weedy Rice as the First Step of Domestication towards Direct-Seeding Rice Using CRISPR/Cas9-Targeted Mutagenesis

With the scarcity of fresh water resources and the increase in labor prices, rice cultivation is changing from transplanting to direct seeding. Weedy rice is a malignant weed with strong drought tolerance in seed germination in direct-seeding rice fields. As the same species of cultivated rice (Oryza sativa L.), weedy rice (Oryza sativa f. spontanea) has the potential of domestication into direct-seeding rice with strong drought tolerance in seed germination by changing a few unfavorable traits. The red pericarp, one of the harmful traits of weedy rice, seriously affects the quality and commercial value of cultivated rice. The recently developed CRISPR/Cas9 (Clustered Regular Interspaced Short Palindromic Repeats) technology can accurately edit the function of genes, providing a possibility for the directed evolution. Weedy rice (WRL-162) with red pericarp was used as a mutant material. Two mutation sites, upstream and downstream of the 14 bp differential fragment of exon 6 of Rc gene, were designed using the CRISPR-Cas9 gene-editing technique. Consequently, three mutant types in T1 generation weedy rice with CRISPR-Cas9 vector changed from red to white pericarp. One mutant type in T1 generation weedy rice with CRISPR-Cas9 vector maintained red pericarp. Comparing the wild type of weedy rice and rice variety control (Nipponbare), mutant types in T3 generation weedy rice without CRISPR-Cas9 vector significantly improved the drought resistance in seed germination. In addition, there was no significant difference in panicle number, seed setting rate per plant, grain length and width between wild type and T3 generation mutant weedy rice without CRISPR-Cas9 vector. Comparing the wild type, some mutant types in T3 generation weedy rice without CRISPR-Cas9 vector significantly decreased plant height, decreased spikelet number per main panicle, decreased plump seed number per main panicle, and increased 1000-grain weight. Our results showed that the Rc gene editing using CRISPR/Cas9 technology can not only remove the harmful pericarp character of weedy rice, but also improve drought tolerance in seed germination. This study might provide new insights for the utilization of weedy rice as germplasm resources for direct-seeding rice by precisely editing genes to remove unfavorable traits.

CRISPR-Cas-Based Epigenome Editing as a Novel Therapeutic Strategy for Metabolic Disorders: Targeting Gene Expression Patterns in Specific Tissues to Treat Diabetes and Obesity

Metabolic disorders such as diabetes and obesity are major public health concerns that affect millions of people worldwide. Epigenetic modifications have been implicated in the development and progression of these disorders, making them attractive targets for therapeutic intervention. The emergence of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPRCas-based) epigenome editing technology has revolutionized the field of gene editing and holds great promise for the treatment of metabolic disorders. However, there are still significant challenges and ethical concerns that must be addressed before this technology can be safely and effectively used in clinical applications. This review provides an overview of epigenetic modifications and their role in metabolic disorders, as well as an explanation of CRISPR-Cas-based epigenome editing and its potential applications. The current state of research and major findings on using this technology for the treatment of metabolic disorders are discussed, along with the advantages and limitations of different delivery strategies. The potential for off-target effects and unintended consequences, as well as strategies for minimizing or mitigating these effects, are also considered. Ethical and regulatory issues associated with CRISPR-Cas-based epigenome editing for metabolic disorders are discussed, with a focus on the importance of responsible development and implementation of this technology. The potential societal impacts of CRISPR-Cas-based epigenome editing for metabolic disorders are also considered. In conclusion, this review highlights the potential of CRISPR-Cas-based epigenome editing as a therapeutic strategy for metabolic disorders, but also underscores the importance of rigorous safety and efficacy testing and responsible implementation. Ultimately, the success of this technology will depend on a balanced consideration of the scientific, ethical, and societal implications.

LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2.

Simple SummaryIn this study, we aimed to establish a visual, rapid, low-cost, sensitive, specific, and portable nucleic acid detection method for PCV2 through coupling LAMP with CRISPR/Cas12a. All the results of LAMP-CRISPR detection, including a low detectable limit of 1 copy/μL, no cross-reaction with main porcine DNA or RNA viruses, and a 100% coincidence rate with qPCR detection, demonstrated that this method was reliable. It has laid the foundation for developing a PCV2 detection kit based on this LAMP-CRISPR method.Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highly sensitive, on-site, and visual diagnostic approach would facilitate dealing with the spread of PCV2. In this study, we intended to establish a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). Following a series of optimizations of its reaction conditions, this LAMP-CRISPR-based PCV2 detection could be conducted in constant temperature equipment, with the result reflected in a direct visual readout way. This established PCV2 detection approach presented fine sensitivity, rapidity, specificity, and reliability, as demonstrated by a low detectable limit of 1 copy/μL, completed within an hour, no cross-reaction with main porcine DNA or RNA viruses like PCV1, PCV3, and PEDV, and a 100% coincidence rate with that of the quantitative PCR (qPCR) method in the evaluation of 30 clinical blood samples, respectively. Therefore, this novel method makes rapid, on-site, visual, highly sensitive, and specific detection of PCV2 possible, facilitating the prevention of this pathogen in the field.

CRISPR-OTE: Prediction of CRISPR On-Target Efficiency Based on Multi-Dimensional Feature Fusion

ObjectiveClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a powerful genome editing technology. Guide RNA (gRNA) plays an essential guiding role in the CRISPR system by complementary base pairing with target DNA. Since the CRISPR targeting mechanism problem has not yet been fully resolved, it remains a challenge to predict gRNA on-target efficiency. Current gRNA design tools often lack efficient information extraction and cannot learn the target efficiency patterns thoroughly. Material and methodsIn this study, CRISPR-OTE is proposed to consider both multi-dimensional sequence information and important complementary prior knowledge based on a simple but effective framework. CRISPR-OTE consists of the local-contextual information branch and the prior knowledge branch. The local-contextual information branch extracts multi-dimensional sequence features from the DNA primary sequence by a parallel framework of Convolutional Neural Networks (CNN) and bidirectional Long Short-Term Memory networks (biLSTM). The prior knowledge branch selects the optimal subset of physicochemical features to provide the neural network with complementary knowledge, such as complex secondary structures. A simple feature fusion strategy is also adopted to fully utilize multi-modal data from the two branches. ResultsThe experimental results show that the optimal subset of physicochemical features (RNA secondary structure and melting temperature of 34nt target) can effectively improve the prediction performance. Additionally, combining multi-dimensional sequence features and multi-modal features can extract information more comprehensively. Through transfer learning, CRISPR-OTE trained on the CRISPR-Cpf1 system can also be successfully applied to the CRISPR-Cas9 system. ConclusionThe performance of CRISPR-OTE is superior to other methods in different CRISPR systems and species. Therefore, CRISPR-OTE is a simple on-target efficiency prediction framework with better accuracy and generalization performance.

The Evolution of CRISPR Technology from Editing to Diagnostics

The Evolution of CRISPR Technology from Editing to Diagnostics

CRISPR-Based Genome-Editing Tools for Huntington's Disease Research and Therapy.

Huntington's disease (HD) is an autosomal dominantly-inherited neurodegenerative disease, which is caused by CAG trinucleotide expansion in exon 1 of the Huntingtin (HTT) gene. Although HD is a rare disease, its monogenic nature makes it an ideal model in which to understand pathogenic mechanisms and to develop therapeutic strategies for neurodegenerative diseases. Clustered regularly-interspaced short palindromic repeats (CRISPR) is the latest technology for genome editing. Being simple to use and highly efficient, CRISPR-based genome-editing tools are rapidly gaining popularity in biomedical research and opening up new avenues for disease treatment. Here, we review the development of CRISPR-based genome-editing tools and their applications in HD research to offer a translational perspective on advancing the genome-editing technology to HD treatment.

GmMDE genes bridge the maturity gene E1 and florigens in photoperiodic regulation of flowering in soybean.

Soybean (Glycine max) is highly sensitive to photoperiod, which affects flowering time and plant architecture and thus limits the distribution range of elite soybean cultivars. The major maturity gene E1 confers the most prominent effect on photoperiod sensitivity, but its downstream signaling pathway remains largely unknown. Here, we confirm that the encoded E1 protein is a transcriptional repressor. The expression of seven GmMDE genes (Glycine max MADS-box genes downregulated by E1) was suppressed when E1 was overexpressed and promoted when E1 was knocked out through clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis. These GmMDEs exhibited similar tissue specificity and expression patterns, including in response to photoperiod, E1 expression, and E1 genotype. E1 repressed GmMDE promoter activity. Results for two GmMDEs showed that E1 epigenetically silences their expression by directly binding to their promoters to increase H3K27me3 levels. The overexpression of GmMDE06 promoted flowering and post-flowering termination of stem growth. The late flowering phenotype of E1-overexpressing soybean lines was reversed by the overexpression of GmMDE06, placing GmMDE06 downstream of E1. The overexpression of GmMDE06 increased the expression of the soybean FLOWERING LOCUS T orthologs GmFT2a and GmFT5a, leading to feedback upregulation of GmMDE, indicating that GmMDE and GmFT2a/GmFT5a form a positive regulatory feedback loop promoting flowering. GmMDE06 also promoted post-flowering termination of stem growth by repressing the expression of the shoot identity gene Dt1. The E1-GmMDEs-GmFT2a/5a-Dt1 signaling pathway illustrates how soybean responds to photoperiod by modulating flowering time and post-flowering stem termination.

The Central Dogma revisited: Insights from protein synthesis, CRISPR, and beyond.

Francis Crick advanced two distinct but interrelated fundamental principles of molecular biology: (1) the Sequence Hypothesis and (2) the Central Dogma. The Sequence Hypothesis defines biological information transfer as the residue-by-residue transfer of sequence information between nucleic acids and to proteins. This is commonly summarized as DNA➔RNA➔protein and is colloquially referred to as the Central Dogma. More specifically, however, the Central Dogma expounded by Crick included a critical restriction, stipulating that "once sequential information has passed into protein it cannot get out again." Under this definition, the Central Dogma has stood the test of time despite challenges. In principle, a violation of the Central Dogma could transpire through synthetic biology or by natural occurrence. To address these possibilities, we draw insights from existing modes of information transfer in protein synthesis and from synthetic Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) gene-editing. We introduce a three-part evaluation scheme, which we apply to the CRISPR/Cas9 system and the more recent CRISPR prime editing system. Potential mechanisms by which engineered sequence editing systems might violate the Central Dogma are considered. We conclude that although information transfer in protein synthesis and CRISPR gene-editing remain within the bounds of the Central Dogma, the underlying mechanisms point toward an avenue of synthetic biology that could directly violate the Central Dogma. Finally, we speculate on some of the theoretical and practical implications of a protein-derived information transfer system. This article is categorized under: RNA Evolution and Genomics > Ribonomics RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Mechanisms.

Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32.

BackgroundOne of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples.Methodology/Principal findingsA Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4–6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study.Conclusions/SignificanceThe RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

Effect of Gene Editing on Alzheimer's Disease

Abstract Background Alzheimer’s disease is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and, eventually, the ability to carry out the simplest tasks. In most people with Alzheimer’s, symptoms first appear in their mid-60s. Current estimates suggest that 44 million people live with dementia worldwide at present. This is predicted to more than triple by 2050 as the population ages. Alzheimer’s disease is currently ranked as the sixth leading cause of death in the United States, but recent estimates indicate that the disorder may rank third, just behind heart disease and cancer, as a cause of death for older people Treatment is currently targeted toward symptomatic therapy, although trials are underway that aim to reduce the production and overall burden of pathology within the brain. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has shown promise in certain neurological disorders, it provides a precise editing to human genome and reflect an efficient curative therapy. We aimed to investigate the efficacy of knock-out of the APP gene “Amyloid precursor protein gene(APBA2)that consequently modify the expression of Amyloid protein in leucocytes cell line using CRISPR-cas9 technology. Methods The gene expression profile of Alzheimer's disease was downloaded from biological bioinformatics databases,and based on bioinformatics analysis, we figured out that APP gene was overexpressed in Alzheimer's disease in both brain and peripheral tissues such as plasma, fibroblast and PMNLs. We used PMNLs as the source of gene for edition in our study .We knocked out the APP gene in leucocytes cell lines using CRISPR-cas9 technology. Finally, the gene editing efficacy was evaluated by cell viability assay, the gene expression was measured by qPCR and the Amyloid protein expression was proved by Immunofluorescence. Results knockout of APP gene int Leucocytes Cell line resulted in reduction in cell viability that was associated with marked reduction in the amyloid protein and gene expressions. Conclusion knockout of APP(APBA2) gene represents a promising therapeutic strategy in Alzheimer's disease.

Comparison of Conventional Molecular and Whole-Genome Sequencing Methods for Differentiating Salmonella enterica Serovar Schwarzengrund Isolates Obtained from Food and Animal Sources.

Over the last decade, Salmonella enterica serovar Schwarzengrund has become more prevalent in Asia, Europe, and the US with the simultaneous emergence of multidrug-resistant isolates. As these pathogens are responsible for many sporadic illnesses and chronic complications, as well as outbreaks over many countries, improved surveillance is urgently needed. For 20 years, pulsed-field gel electrophoresis (PFGE) has been the gold standard for determining bacterial relatedness by targeting genome-wide restriction enzyme polymorphisms. Despite its utility, recent studies have reported that PFGE results correlate poorly with that of closely related outbreak strains and clonally dominant endemic strains. Due to these concerns, alternative amplification-based molecular methods for bacterial strain typing have been developed, including clustered regular interspaced short palindromic repeats (CRISPR) and multilocus sequence typing (MLST). Furthermore, as the cost of sequencing continues to decrease, whole genome sequencing (WGS) is poised to replace other molecular strain typing methods. In this study, we assessed the discriminatory power of PFGE, CRISPR, MLST, and WGS methods to differentiate between 23 epidemiologically unrelated S. enterica serovar Schwarzengrund isolates collected over an 18-year period from distinct locations in Taiwan. The discriminatory index (DI) of each method for different isolates was calculated, resulting in values between 0 (not discriminatory) and 1 (highly discriminatory). Our results showed that WGS has the greatest resolution (DI = 0.982) compared to PFGE (DI = 0.938), CRISPR (DI = 0.906), and MLST (DI = 0.463) methods. In conclusion, the WGS typing approach was shown to be the most sensitive for S. enterica serovar Schwarzengrund fingerprinting.

Genome Editing of the Murine Calreticulin Gene Using the CRISPR/Cas9 and CPF1 System

<Background> Abnormalities of the Calreticulin (CALR) gene are frequently observed in human Myeloproliferative Neoplasms (MPN). Small deletions and insertions in the last exon of CALR, leading to frame-shift mutations, are common genetic alterations in this disease. Genome editing by the CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats and CRISPR-Associated proteins 9) system is one of ways to manipulate genes in normal/abnormal mammalian cells. Recently, CPF1 (CRISPR from Prevotella and Francisella 1) which is a novel single-component RNA-guided endonuclease has been identified. CPF1 cleaves double-strand DNA with a 5' overhang protruding end while Cas9 cleaves DNA with a blunt end. We have manipulated the murine calreticulin gene (calr) to introduce similar genetic defects as observed in human MPN using Cas9 and two kinds of CFP1 enzymes (AsCPF1 and LbCPF1). Efficiency of genetic alterations by the Cas9, AsCPF1 and LbCPF1 was compared. Furthermore, we used these enzymes to create Knock-In (KI) cells carrying an artificial exons in the last exon of calr .<Methods> Two guide RNAs targeting the last exon of calr were designed for Cas9, AsCPF1 and LbCPF1. Vectors encoding the Cas9 or CPF1 nuclease with GFP/YFP as a marker were transfected into murine fibroblast cell line, NIH3T3, with the vectors encoding the guide RNAs. Transfected cells were sorted on a flow cytometer using the GFP/YFP as the marker. Single cell culture was performed in 96-well plates. Six, 11, 10 clones for Cas9, AsCPF1, LbCPF1 were isolated and nucleotide sequences of the last exon of calr were confirmed by direct sequencing. Expression of the transcripts from the altered alleles were also confirmed by reverse-transcription(RT) PCR and sequencing. Furthermore, we have created a KI construct in which the last exon of calr was divided into three artificial exons and the middle exon was flanked by LoxP sequences (floxed). The KI construct was tranfected into NIH3T3 with vectors encoding the Cas9 or CFP1 as well as vectors encoding the guide RNAs. Single clones were isolated and genetic status of the last exon of calr in each clone was examined by PCR/sequencing.<Results> Nine of 11 clones for AsCPF1 (82%) and nine of 10 clones for LbCPF1 (90%) showed genetic abnormalities in the last exon of calr . Cas9 showed five manipulated clones of six clones (83%). Two, Three and Three clones were hemizygous abnormalities for Cas9, AsCPF1 and LbCPF1 respectively (33%, 27% and 30%). Three, four and four clones were compound-heterozygous abnormalities for Cas9, AsCPF1 and LbCPF1 respectively (50%, 36% and 40%). One clone for AsCPF1 and one clone for LbCPF1 were homozygous abnormalities. Majority of the genetic abnormalities were small deletion (1- 55bp). Three clones for Cas9 showed complete removal of the genome between the two target sites (168-170bp). One clone for Cas9 showed 163bp insertion in the target region. Altered messages transcribed from the manipulated alleles were confirmed by RT-PCR as well as sequencing in all the clones. As for the experiments to create the KI cells, more than 50 clones were isolated and one clone showed replacement of the last exon of calr by the KI construct. Transient expression of the Cre recommbinase by transfection efficiently deleted the floxed exon. However, we found that the KI cells expressed altered calr messages with complex sequence due to an unintentional insertion of the DNA fragment in the artificial exons.<Discussion> With the Cas9, AsCPF1 and the LbCPF1 system, we successfully introduced genetic changes of calr in murine cell line. The efficacy of introducing genomic abnormalities into calr was similar among the three endonucleases, Cas9, AsCPF1, and LbCPF1. Small deletions in calr were efficiently introduced in the cell line. However, efficiency of KI was not sufficient. Optimization may be needed to create KI cell lines using the CRISPR/Cas9 system. Nevertheless, this technique can be applied for generation of murine models carrying the pathogenic calr allele or artificial conditional exons of calr . DisclosuresNo relevant conflicts of interest to declare.

CRISPR/Cas9 mediated knock-out of VPREB1 gene induces a cytotoxic effect in myeloma cells.

BackgroundMultiple Myeloma (MM) is a heterogeneous, hematological neoplasm that accounts 2% of all cancers. Although, autologous stem cell transplantation and chemotherapy are currently the most effective therapy, it carries a notable hazards, in addition for being non curative. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has been successfully tried at the experimental level, for the treatment of several hematological malignancies.ObjectivesWe aimed to investigate the in-vitro effect of CRISPR-cas9-mediated knock-out of V-set pre B-cell surrogate light chain 1”VPREB1” gene on the malignant proliferation of primary cultured myeloma cells.MethodsBioinformatics’ analysis was performed to explore the gene expression profile of MM, and the VPREB1 gene was selected as a target gene for this study. We knocked-out the VPREB1 gene in primary cultured myeloma cells using CRISPR-cas9, the VPREB1 gene editing efficacy was verified by determining VPREB1 gene expression at both the mRNA and protein levels by qPCR and immunofluorescence, respectively. Furthermore, the cytotoxic effect on primary myeloma cells proliferation was evaluated using cytotoxicity assay.ResultsThere was a statistically significant reduction of both VPREB1 mRNA and protein expression levels (p<0.01). knock-out of VPREB1 gene in myeloma cell line resulted in a statistically significant reduction of myeloma cell proliferation.ConclusionCRISPR-cas9-mediated knock-out of VPREB1 gene is effective for inhibiting the proliferation of primary myeloma cells. This would provide a basis for a promising therapeutic strategy for patients with multiple myeloma.

Generation of a novel decay accelerating factor (DAF) knock-out rat model using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9), genome editing.

Decay accelerating factor (DAF), a key complement activation control protein, is a 70kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf-/-) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf+/+ rats. Renal histology in Daf-/- rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf+/+ rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf-/- rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.

COVID-19 one year later: a retrospect of CRISPR-Cas system in combating COVID-19.

Coronavirus disease 2019 (COVID-19), an infectious disease caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a persistent global threat. The transmission of SARS-CoV-2 is wide and swift. Rapid detection of the viral RNA and effective therapy are imperative to prevent the worldwide spread of the new infectious disease. Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein (Cas) system is an RNA-directed adaptive immune system, and it has been transformed into a gene editing tool. Applications of CRISPR-Cas system involves in many fields, such as human gene therapy, drug discovery and disease diagnosis. Under the background of COVID-19 pandemic, CRISPR-Cas system shows hidden capacity to fight the emergency in many aspects. This review will focus on the role of gene editing in COVID-19 diagnosis and treatment. We will describe the potential use of CRISPR-Cas-based system in combating COVID-19, from diagnosis to treatment. Furthermore, the limitation and perspectives of this novel technology are also evaluated.

NEED OF OUR TIMES: IMPACT OF GENOME EDITING SCIENCES AND REQUISITE PREPAREDNESS FOR MOLECULAR BIOTERRORISM

Not Applicable.