CRISPR-associated Protein Research Articles

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

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

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

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

A simple, cost-effective, and efficient method for screening CRISPR/Cas9 mutants in plants

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing system is widely used for targeted mutagenesis in a growing number of plant species. To streamline the screening process for mutants, especially those generated from low-efficiency editing events, there is a need for a rapid, cost-effective, and efficient method. Although several screening methods have been developed to process initial samples, these methods often tend to be time-consuming, expensive, or inefficient when dealing with larger sample sizes. Here we describe a simple, rapid, low-cost, and sensitive screening method for screening CRISPR/Cas9 mutants called PCR-Bsl I-associated analysis (PCR-BAA). This method requires only standard PCR and Bsl I restriction enzyme digestion, as well as agarose gel electrophoresis analysis. This method is particularly well suited for the efficient screening of mutants from larger populations of transformants. The simplicity, low cost, and high sensitivity of the PCR-BAA method make it particularly suitable for rapid screening of CRISPR/Cas9-induced mutants, especially those from low-efficiency editing events.

CRISPR-Cas9: Revolutionizing Gene Therapy for Genetic Disorders

Faulty or mutated genes cause multiple human disorders such as cancer, neurogenerative disorders, and cardiovascular diseases. These mutations in genes are usually inherited. Many treatments have been established to cure such diseases, but gene therapy is the most promising strategy. It’s an application of biotechnology that is based on correcting and replacing the mutated gene with the healthy gene, providing the genes for the proper expression of desired proteins required for curing the disease. In clinical settings, gene therapy has proved itself as a very promising treatment but has some drawbacks. Traditionally, these methods involved viral vectors that are used to deliver correct genes and replace them with the mutated genes, in patients. These approaches have somehow potential to cure diseases but often off-target effects, limitations in the editing process, and immune responses caused by patients’ immune systems are the main challenges that are the main shortcomings of this method. However, the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease protein 9 (Cas9) genome editing system (CRISPR/Cas9) in 2012 and its development have increased the value of gene therapy in the therapeutic world [1]. CRISPR/Cas9 is a revolutionizing tool that has been in gene therapy for knocking in and out gene to correct the mutation associated with many genetic diseases. This system has evolved greatly and has many isomers using different strategies to improve both applied, basic research and its clinical application. The ability of CRISPR-Cas9 to target various genetic diseases is one of its greatest advantages. Mutation in single gene results in monogenic diseases such as cystic fibrosis, muscular dystrophy, and sickle cell anemia, and this system has shown the potential to edit or fix the defective genes, causing these diseases. Even in the case of complex disorders caused by the mutation in multiple genes such as several types of cancer, and cardiovascular diseases, CRISPR has shown very positive results. The application of CRISPR-Cas9 in gene therapy is very promising and has tremendous potential to give cures for many complex disorders. The ethical issue regarding the editing of human DNA and the inheritance of modified DNA into the next generation is still under discussion and causing hurdles in uncovering the full potential of this system. Researchers are working to increase its efficiency and specificity to reduce the off-target sequences so that only targeted modification can be achieved. Moreover, they are trying to give possible results to reduce ethical concerns. CRISPR has evolved greatly and has many isomers using different strategies to improve both applied, basic research and its clinical application in the future.

CRISPR-Cas9 and its Application in the Fields of Medics

Gene editing technology has become a prominent objective within the fields of medical research, the clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is one particular technology that has already been practiced in a set of clinical trials, which produced a satisfying result that have demonstrated its potential to be applied to a great range of treatments. CRISPR-Cas9 was derived from bacterial defense system, and has been applied in the field of medical research. For instance, the research practice of using CAR-T cells by CAR-T Therapy, and genetically modifying DNA strands to remove cancerous or mutated sequences. This research has great chance to promote treatments associated with genetic modification and provide new hope for patients in the future. Nevertheless, this technology is still immature, due to its potential drawbacks. Most researchers are still focusing on practices of CRISPR-Cas9 on animal models or isolated somatic cells. This article draws attention on different treatments of CRISPR-Cas9 and provides evaluation and suggestions as a reference for future practices of this technology. Overall, there are two discoveries of advancement to the CRISPR-Cas9 mechanism: modification to the guide RNA that improves the accuracy, and applying high-fidelity Cas9 protein variants to enhance its comprehensive accuracy.

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.

Characterization of NFDQ1 in Cryptosporidium parvum

BackgroundCryptosporidium spp. are important zoonotic parasites that can cause moderate to severe diarrhea in humans and animals. Among the three Cryptosporidium species infecting the intestines of calves, Cryptosporidium parvum has a broad host range and causes severe diarrhea in calves, while Cryptosporidium bovis and Cryptosporidium ryanae mainly infect calves without obvious clinical symptoms. Comparative genomic analysis revealed differences in the copy number of genes encoding the nonfinancial disclosure quality (NFDQ) secretory protein family among the three species, suggesting that this protein family may be associated with the host range or pathogenicity of Cryptosporidium spp. To understand the function of cgd8_10 encoded NFDQ1, tagged and knockout strains were constructed and characterized in this study.MethodsTo determine the localization of NFDQ1, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to tag the C-terminus of NFDQ1 with three hemagglutinin epitopes (3 × HA). The tagged strain was constructed, and the genomic insertion was confirmed by polymerase chain reaction (PCR). Immunofluorescence assays were performed to observe the localization of NFDQ1 both in extracellular sporozoites and at various intracellular developmental stages. Immunoelectron microscopy was used to study the ultrastructural localization of NFDQ1. Then, the ΔNFDQ1 strain was generated by CRISPR/Cas9 and the in vitro growth assay on HCT-8 cells was used to analyze of phenotypic changes after knockout NFDQ1 in parasites.ResultsThe NFDQ1 tagging and knockout stains were successfully constructed by CRISPR/Cas9 technology and the insertions of transgenic strains were validated by PCR. The expression of NFDQ1 was validated in parasite by western blot. Immunofluorescence and immune-electron microscopy assay showed that NFDQ1 expressed in both asexual and sexual stages of C. parvum, where it was localized to the cytoplasm of the parasite. Upon ablation of NFDQ1, the ΔNFDQ1 strain showed an apparent growth retardation during sexual replication in vitro.ConclusionsNFDQ1 is a cytoplasmic protein without specific localization to secretory organelles, and it may participate in C. parvum growth during sexual reproduction. Future study should determine the role of NFDQ1 following C. parvum infection in vivo.Graphical

CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing system has altered plant research by allowing for targeted genome alteration, and they are emerging as powerful tools for evaluating plant gene function and improving crop yield. Even though CRISPR/Cas9 cleavage and subsequent repair are effective ways to precisely replace genes and change base pairs in plants, the dominance of the non-homologous end-joining pathway (NHEJ) and homology-directed repair's (HDR) poor effectiveness in plant cells have restricted their use. Base editing is gaining popularity as a potential alternative to HDR or NHEJ-mediated replacement, allowing for precise changes in the plant genome via programmed conversion of a single base to another without the need for a donor repair template or double-stranded breaks. In this review, we primarily present the mechanisms of base-editing system, including their distinct types such as DNA base editors (cytidine base editor and adenine base editor) and RNA base editors discovered so far. Next, we outline the current potential applications of the base-editing system for crop improvements. Finally, we discuss the limitations and potential future directions of the base-editing system in terms of improving crop quality. We hope that this review will enable the researcher to gain knowledge about base-editing tools and their potential applications in crop improvement.

CRISPR-Based Therapy for Hereditary Angioedema.

Hereditary angioedema is a rare genetic disease characterized by severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy that is based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1). A single dose of NTLA-2002 may provide lifelong control of angioedema attacks. In this phase 2 portion of a phase 1-2 trial, we randomly assigned adults with hereditary angioedema in a 2:2:1 ratio to receive NTLA-2002 in a single dose of 25 mg or 50 mg or placebo. The primary end point was the number of angioedema attacks per month (the monthly attack rate) from week 1 through week 16. Secondary end points included safety, pharmacokinetics, and pharmacodynamics (i.e., the change from baseline in total plasma kallikrein protein level); exploratory end points included patient-reported outcomes. Of the 27 patients who underwent randomization, 10 received 25 mg of NTLA-2002, 11 received 50 mg, and 6 received placebo. From week 1 through week 16, the estimated mean monthly attack rate was 0.70 (95% confidence interval [CI], 0.25 to 1.98) with 25 mg of NTLA-2002, 0.65 (95% CI, 0.24 to 1.76) with 50 mg, and 2.82 (95% CI, 0.80 to 9.89) with placebo; the difference in the estimated mean attack rate with NTLA-2002 as compared with placebo was -75% with 25 mg and -77% with 50 mg. Among patients who received NTLA-2002, 4 of the 10 patients who received 25 mg (40%) and 8 of the 11 who received 50 mg (73%) were attack-free with no additional treatment during the period from week 1 through week 16. The most common adverse events among patients who received NTLA-2002 were headache, fatigue, and nasopharyngitis. The mean percent change in total plasma kallikrein protein levels from baseline to week 16 was -55% with 25 mg and -86% with 50 mg; levels remained unchanged with placebo. NTLA-2002 administered in a single dose of 25 mg or 50 mg reduced angioedema attacks and led to robust and sustained reduction in total plasma kallikrein levels in patients with hereditary angioedema. These results support continued investigation in a larger phase 3 trial. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830; EudraCT number, 2021-001693-33.).

Genome Editing for Grass Improvement and Future Agriculture

Abstract Grasses, including turf and forage, cover most of the earth’s surface; predominantly important for land, water, livestock feed, soil and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.

SlMKK4 is responsible for pollen development in tomato

The development of viable pollen is a determinant of male fertility and plays an essential role in the reproductive process of angiosperms. Mitogen-activated protein kinase (MAPK) cascades modulate diverse aspects of plant growth, but their involvement in post-meiotic pollen development is unclear. In this study, SlMKK4 was identified as a crucial regulator in overseeing pollen development in tomatoes (Solanum lycopersicum). Utilizing CRISPR-associated protein 9 to disrupt SlMKK4 resulted in an obvious decrease in pollen viability. The results of cell biology and transcriptomic analyses demonstrated that SlMKK4 specifically regulates auxin and sugar metabolism as well as signal transduction during post-meiotic pollen development. This is supported by the finding that protein–protein interaction assays and in vitro phosphorylation assays indicate that SlMKK4 serves as the upstream MAPKK for SlMPK20, which exhibits a distinct function in regulating the uninucleate (UN) to binucleate (BN) transition during microgametogenesis in tomatoes. Moreover, pollen from transgenic plants experienced significant arrest predominantly at the BN stage, accompanied by subcellular abnormalities manifesting during the late UN microspore phase. Furthermore, transcriptomic analyses indicated that SlMKK4 knockout remarkably downregulated the expression of numerous genes regulating auxin and sugar metabolism as well as signal transduction in anthers. Therefore, our findings suggest that SlMKK4 may serve as one of the upstream SlMAPKKs of SlMPK20 and also play a pivotal role in modulating post-meiotic pollen development in tomato plants.

Recent advances and current status of gene therapy for epilepsy.

Epilepsy is a common neurological disorder with complex pathogenic mechanisms, and refractory epilepsy often lacks effective treatments. Gene therapy is a promising therapeutic option, with various preclinical experiments achieving positive results, some of which have progressed to clinical studies. This narrative review was conducted by searching for papers published in PubMed/MEDLINE with the following single and/or combination keywords: epilepsy, children, neurodevelopmental disorders, genetics, gene therapy, vectors, transgenes, receptors, ion channels, micro RNAs (miRNAs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 (CRISPR/Cas9), expression regulation, optogenetics, chemical genetics, mitochondrial epilepsy, challenges, ethics, and disease models. Currently, gene therapy research in epilepsy primarily focuses on symptoms attenuationmediated by viral vectors such as adeno-associated virus and other types. Advances in gene therapy technologies, such as CRISPR/Cas9, have provided a new direction for epilepsy treatment. However, the clinical application still faces several challenges, including issues related to vectors, models, expression controllability, and ethical considerations. Here, we summarize the relevant research and clinical advances in gene therapy for epilepsy and outline the challenges facing its clinical application. In addition to the shortcomings inherent in gene therapy components, the reconfiguration of excitatory and inhibitory properties in epilepsy treatment is a delicate process. On-demand, cell-autonomous treatments and multidisciplinary collaborations may be crucial in addressing these issues. Understanding gene therapy for epilepsy will help clinicians gain a clearer perception of the research progress and challenges, guiding the design of future clinical protocols and research decisions.

Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

Future prospects for the advancement of treatment of men with NOA: focus on gene editing, artificial sperm, stem cells, and use of imaging.

Nonobstructive azoospermia (NOA) affects about 60% of men with azoospermia, representing a severe form of male infertility. The current approach to manage NOA primarily involves testicular sperm retrieval methods such as conventional testicular sperm extraction (c-TESE) and microdissection testicular sperm extraction (micro-TESE). While combining testicular sperm retrieval with intracytoplasmic sperm injection (ICSI) offers hope for patients, the overall sperm retrieval rate (SRR) stands at around 50%. In cases where micro-TESE fails to retrieve sperm, limited options, like donor sperm or adoption, can be problematic in certain cultural contexts. This paper delves into prospective treatments for NOA management. Gene editing technologies, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 (CRISPR/Cas9), hold potential for correcting genetic mutations underlying testicular dysfunction. However, these technologies face challenges due to their complexity, potential off-target effects, ethical concerns, and affordability. This calls for research to address key challenges associated with NOA management within the clinical settings. This also necessitate ongoing research essential for developing more sensitive diagnostic tests, validating novel treatments, and customizing current treatment strategies for individual patients. This review concluded that the future of NOA management may entail a combination of these treatment options, tailored to each patient's unique circumstances, providing a comprehensive approach to address NOA challenges.

Genome Editing in Apicomplexan Parasites: Current Status, Challenges, and Future Possibilities.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has revolutionized genome editing across various biological systems, including the Apicomplexa phylum. This review describes the status, challenges, and applications of CRISPR-Cas9 editing technology in apicomplexan parasites, such as Plasmodium, Toxoplasma, Theileria, Babesia, and Cryptosporidium. The discussion encompasses successfully implemented CRISPR-Cas9-based techniques in these parasites, highlighting the achieved milestones, from precise gene modifications to genome-wide screening. In addition, the review addresses the challenges hampering efficient genome editing, including the parasites' complex life cycles, multiple intracellular stages, and the lack of robust genetic tools. It further explores the ethical and policy considerations surrounding genome editing and the future perspectives of CRISPR-Cas applications in apicomplexan parasites.

Reverse-transcription recombinase-aided amplification and CRISPR/Cas12a-based universal detection for fast screening and accurate identification of six pospiviroids infecting Solanaceae crops.

Pospiviroids, members of the genus Pospiviroid, can cause severe diseases in tomato and other Solanaceae crops, causing considerable economic losses worldwide. Six pospiviroids including potato spindle tuber viroid (PSTVd), tomato chlorotic dwarf viroid (TCDVd), tomato planta macho viroid (TPMVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), and tomato apical stunt viroid (TASVd) are regulated in many countries and organizations. Rapid, accurate detection is thus crucial for controlling the spread of these pospiviroids. For simultaneous detection of these six pospiviroids, we developed a rapid, visual method that uses a reverse transcription recombinase-aided amplification (RT-RAA) assay coupled with a clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 12a (CRISPR/Cas12a) system. In particular, this technique could achieve both universal detection and specific identification of the six target pospiviroids within 40 min. The universal detection could diagnose the six target pospiviroids in a single reaction, and the specific identification could identify each target pospiviroid without cross-reactivity of other pospiviroids. The sensitivity limits for the target pospiviroids detection with the proposed detection method were higher than those of the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. We designed an RT-RAA-CRISPR/Cas12a-based universal detection method for both large-scale screening and accurate identification of the six target pospiviroids, which is appropriate for on-site detection. Our study results can aid in performing rapid, large-scale screening of multiple pests simultaneously. © 2024 Society of Chemical Industry.

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication.

Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA sequencing (RNA-seq) analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range downregulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.

Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.

Exploring retinal degenerative diseases through CRISPR-based screening.

The CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein9) system has emerged as a powerful genetic tool, gaining global recognition as a versatile and efficient gene-editing technique. Its transformation into a high-throughput research platform, CRISPR Screening, has demonstrated wide applicability across various fields such as cancer biology, virology, and drug target discovery, resulting in significant advances. However, its potential in studying retinal degenerative diseases remains largely unexplored, despite the urgent need for effective treatments arising from an incomplete understanding of disease mechanisms. This review aims to present a comprehensive overview of the evolution and current state of CRISPR tools and CRISPR screening methodologies. Noteworthy pioneering studies utilizing these technologies are discussed, alongside experimental design guidelines, including positive and negative selection strategies and delivery methods for sgRNAs (single guide RNAs) and Cas proteins. Furthermore, we explore existing in vitro models appropriate for CRISPR screening in retinal research and identify relevant research questions that could be addressed through this approach. It is anticipated that this review will stimulate innovation in retinal research, facilitating a deeper comprehension of retinal pathophysiology and paving the way for groundbreaking therapeutic interventions and enhanced patient outcomes in the management of retinal degenerative disorders.

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

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