Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

Genome wide-scale CRISPR-Cas9 knockout screens identify a fitness score for optimized risk stratification in colorectal cancer.

BACKGROUND: The molecular complexity of colorectal cancer poses a significant challenge to the clinical implementation of accurate risk stratification. There is still an urgent need to find better biomarkers to enhance established risk stratification and guide risk-adapted treatment decisions. METHODS: we systematically analyzed cancer dependencies of 17 colorectal cancer cells and 513 other cancer cells based on genome-scale CRISPR-Cas9 knockout screens to identify colorectal cancer-specific fitness genes. A regression model was built using colorectal cancer-specific fitness genes, which was validated in other three independent cohorts. 30 published gene expression signatures were also retrieved. FINDINGS: We defined a total of 1828 genes that were colorectal cancer-specific fitness genes and identified a 22 colorectal cancer-specific fitness gene (CFG22) score. A high CFG22 score represented unfavorable recurrence and mortality rates, which was validated in three independent cohorts. Combined with age, and TNM stage, the CFG22 model can provide guidance for the prognosis of colorectal cancer patients. Analysis of genomic abnormalities and infiltrating immune cells in the CFG22 risk stratification revealed molecular pathological difference between the subgroups. Besides, drug analysis found that CFG22 high patients were more sensitive to clofibrate. INTERPRETATION: The CFG22 model provided a powerful auxiliary prediction tool for identifying colorectal cancer patients with high recurrence risk and poor prognosis, optimizing precise treatment and improving clinical efficacy.

Adoption of CRISPR-Cas for crop production: present status and future prospects.

Background: Global food systems in recent years have been impacted by some harsh environmental challenges and excessive anthropogenic activities. The increasing levels of both biotic and abiotic stressors have led to a decline in food production, safety, and quality. This has also contributed to a low crop production rate and difficulty in meeting the requirements of the ever-growing population. Several biotic stresses have developed above natural resistance in crops coupled with alarming contamination rates. In particular, the multiple antibiotic resistance in bacteria and some other plant pathogens has been a hot topic over recent years since the food system is often exposed to contamination at each of the farm-to-fork stages. Therefore, a system that prioritizes the safety, quality, and availability of foods is needed to meet the health and dietary preferences of everyone at every time. Methods: This review collected scattered information on food systems and proposes methods for plant disease management. Multiple databases were searched for relevant specialized literature in the field. Particular attention was placed on the genetic methods with special interest in the potentials of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas (CRISPR associated) proteins technology in food systems and security. Results: The review reveals the approaches that have been developed to salvage the problem of food insecurity in an attempt to achieve sustainable agriculture. On crop plants, some systems tend towards either enhancing the systemic resistance or engineering resistant varieties against known pathogens. The CRISPR-Cas technology has become a popular tool for engineering desired genes in living organisms. This review discusses its impact and why it should be considered in the sustainable management, availability, and quality of food systems. Some important roles of CRISPR-Cas have been established concerning conventional and earlier genome editing methods for simultaneous modification of different agronomic traits in crops. Conclusion: Despite the controversies over the safety of the CRISPR-Cas system, its importance has been evident in the engineering of disease- and drought-resistant crop varieties, the improvement of crop yield, and enhancement of food quality.

Strong association between genomic 3D structure and CRISPR cleavage efficiency.

CRISPR is a gene editing technology which enables precise in-vivo genome editing; but its potential is hampered by its relatively low specificity and sensitivity. Improving CRISPR's on-target and off-target effects requires a better understanding of its mechanism and determinants. Here we demonstrate, for the first time, the chromosomal 3D spatial structure's association with CRISPR's cleavage efficiency, and its predictive capabilities. We used high-resolution Hi-C data to estimate the 3D distance between different regions in the human genome and utilized these spatial properties to generate 3D-based features, characterizing each region's density. We evaluated these features based on empirical, in-vivo CRISPR efficiency data and compared them to 425 features used in state-of-the-art models. The 3D features ranked in the top 13% of the features, and significantly improved the predictive power of LASSO and xgboost models trained with these features. The features indicated that sites with lower spatial density demonstrated higher efficiency. Understanding how CRISPR is affected by the 3D DNA structure provides insight into CRISPR's mechanism in general and improves our ability to correctly predict CRISPR's cleavage as well as design sgRNAs for therapeutic and scientific use.

A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library.

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5' end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.

Revolutionizing Lung Cancer Treatment: Innovative CRISPR-Cas9 Delivery Strategies.

Lung carcinoma, including both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), remains a significant global health challenge due to its high morbidity and mortality rates. The objsective of this review is to meticulously examine the current advancements and strategies in the delivery of CRISPR-Cas9 gene-editing technology for the treatment of lung carcinoma. This technology heralds a new era in molecular biology, offering unprecedented precision in genomic modifications. However, its therapeutic potential is contingent upon the development of effective delivery mechanisms that ensure the efficient and specific transport of gene-editing tools to tumor cells. We explore a variety of delivery approaches, such as viral vectors, lipid-based nanoparticles, and physical methods, highlighting their respective advantages, limitations, and recent breakthroughs. This review also delves into the translational and clinical significance of these strategies, discussing preclinical and clinical studies that investigate the feasibility, efficacy, and safety of CRISPR-Cas9 delivery for lung carcinoma. By scrutinizing the landscape of ongoing clinical trials and offering translational perspectives, we aim to elucidate the current state and future directions of this rapidly evolving field. The review is structured to first introduce the problem and significance of lung carcinoma, followed by an overview of CRISPR-Cas9 technology, a detailed examination of delivery strategies, and an analysis of clinical applications and regulatory considerations. Our discussion concludes with future perspectives and challenges, such as optimizing delivery strategies, enhancing specificity, mitigating immunogenicity concerns, and addressing regulatory issues. This comprehensive overview seeks to provide insights into the potential of CRISPR-Cas9 as a revolutionary approach for targeted therapies and personalized medicine in lung carcinoma, emphasizing the importance of delivery strategy development in realizing the full potential of this groundbreaking technology.

DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay.

BACKGROUND: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SIGNIFICANCE: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.

CRISPR/Cas9-induced LEAP2 and GHSR1a knockout mutant zebrafish displayed abnormal growth and impaired lipid metabolism.

Investigating the principles of fish fat deposition and conducting related research are current focal points in fish nutrition. This study explores the endocrine regulation of LEAP2 and GHSR1a in zebrafish by constructing mutantmodels andexamining the effects of the endocrine factors LEAP2 and its receptor GHSR1a on zebrafish growth, feeding, and liver fat deposition. Compared to the wild type (WT), the mutation of LEAP2 results in increased feeding and decreased swimming in zebrafish. The impact is more pronounced in adult female zebrafish, characterized by increased weight, length, width, and accumulation of lipid droplets in the liver.Incontrast, deficiency in GHSR1a significantly reduces the growth of male zebrafish and markedly decreases liver fat deposition.These research findings indicate the crucial roles of LEAP2 and GHSR1a in zebrafish feeding, growth, and intracellular fat metabolism. This study, for the first time, investigated the endocrine metabolic regulation functions of LEAP2 and GHSR1a in the model organism zebrafish, providing initial insights into their effects and potential mechanisms on zebrafish fat metabolism.

Towards a Cure for Diamond-Blackfan Anemia: Views on Gene Therapy.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.

A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.

Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.

Increasing GSH-Px Activity and Activating Wnt Pathway Promote Fine Wool Growth in FGF5-Edited Sheep.

Fibroblast growth factor 5 (FGF5) plays key roles in promoting the transition from the anagen to catagen during the hair follicle cycle. The sheep serves as an excellent model for studying hair growth and is frequently utilized in various research processes related to human skin diseases. We used the CRISPR/Cas9 system to generate four FGF5-edited Dorper sheep and only low levels of FGF5 were detected in the edited sheep. The density of fine wool in GE sheep was markedly increased, and the proportion of fine wool with a diameter of 14.4-20.0 µm was significantly higher. The proliferation signal in the skin of gene-edited (GE) sheep was stronger than in wild-type (WT) sheep. FGF5 editing decreased cortisol concentration in the skin, further activated the activity of antioxidant enzymes such as Glutathione peroxidase (GSH-Px), and regulated the expression of Wnt signaling pathways containing Wnt agonists (Rspondins, Rspos) and antagonists (Notum) in hair regeneration. We suggest that FGF5 not only mediates the activation of antioxidant pathways by cortisol, which constitutes a highly coordinated microenvironment in hair follicle cells, but also influences key signals of the Wnt pathway to regulate secondary hair follicle (SHF) development. Overall, our findings here demonstrate that FGF5 plays a significant role in regulating SHF growth in sheep and potentially serves as a molecular marker of fine wool growth in sheep breeding.

Comparative Evaluation of PCR-Based, LAMP and RPA-CRISPR/Cas12a Assays for the Rapid Detection of Diaporthe aspalathi.

Southern stem canker (SSC) of soybean, attributable to the fungal pathogen Diaporthe aspalathi, results in considerable losses of soybean in the field and has damaged production in several of the main soybean-producing countries worldwide. Early and precise identification of the causal pathogen is imperative for effective disease management. In this study, we performed an RPA-CRISPR/Cas12a, as well as LAMP, PCR and real-time PCR assays to verify and compare their sensitivity, specificity and simplicity and the practicality of the reactions. We screened crRNAs targeting a specific single-copy gene, and optimized the reagent concentrations, incubation temperatures and times for the conventional PCR, real-time PCR, LAMP, RPA and Cas12a cleavage stages for the detection of D. aspalathi. In comparison with the PCR-based assays, two thermostatic detection technologies, LAMP and RPA-CRISPR/Cas12a, led to higher specificity and sensitivity. The sensitivity of the LAMP assay could reach 0.01 ng µL-1 genomic DNA, and was 10 times more sensitive than real-time PCR (0.1 ng µL-1) and 100 times more sensitive than conventional PCR assay (1.0 ng µL-1); the reaction was completed within 1 h. The sensitivity of the RPA-CRISPR/Cas12a assay reached 0.1 ng µL-1 genomic DNA, and was 10 times more sensitive than conventional PCR (1.0 ng µL-1), with a 30 min reaction time. Furthermore, the feasibility of the two thermostatic methods was validated using infected soybean leaf and seeding samples. The rapid, visual one-pot detection assay developed could be operated by non-expert personnel without specialized equipment. This study provides a valuable diagnostic platform for the on-site detection of SSC or for use in resource-limited areas.

A highly specific and ultrasensitive approach to detect Prymnesium parvum based on RPA-CRISPR-LbaCas12a-LFD system.

BACKGROUND: Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance. RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL). SIGNIFICANCE: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.