The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.

Aujeszky's disease (AD) is an acute infectious disease that infects pigs and other animals, resulting in significant economic losses and posing a threat to human health. Reliable and rapid detection methods are essential for the prevention of AD. In this study, a RAA-Cas12b assay based on recombinase-aided amplification (RAA) and CRISPR-Cas12b system was established, optimized and evaluated for the rapid detection of wild-type Pseudorabies Virus (PRV). The results can not only be detected by real-time fluorescence readout, but also can be visualized by a portable blue light instrument. There was no cross-reaction with PRV Bartha-K61 strain or other swine infectious viruses. The analytical sensitivities of the real-time PRV RAA-Cas12b assay and visual PRV RAA-Cas12b assay were determined to be 15 copies/μL with 95 % confidence interval and 140 copies/μL with 95 % confidence interval, respectively. A total of 31 clinical samples were detected and compared with PRV qPCR assay to evaluate the diagnostic performance of the PRV RAA-Cas12b assay. The diagnostic coincidence rate of the two assays was 100 %. In summary, this convenient and reliable assay has great potential for rapid detection of wild type PRV in point-of-care testing (POCT).

This article reviews the mechanisms, advancements, and potential implications of clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) gene editing technology, with a specific focus on its applications in reproductive biology and assisted reproduction. It aims to explore the benefits and challenges of integrating this revolutionary technology into clinical and research settings.

CRISPR-Cas9 is a transformative tool for precise genome editing, enabling targeted modifications through mechanisms like nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Innovations such as Cas9 nickase and dCas9 systems have improved specificity and expanded applications, including gene activation, repression, and epigenetic modifications. In reproductive research, CRISPR has facilitated gene function studies, corrected genetic mutations in animal models, and demonstrated potential in addressing human infertility and hereditary disorders. Emerging applications include mitochondrial genome editing, population control of disease vectors via gene drives, and detailed analyses of epigenetic mechanisms.

CRISPR-Cas9 technology has revolutionized genetic engineering by enabling precise genome modifications. This article discusses its mechanisms, focusing on the repair pathways (NHEJ and HDR) and methods to mitigate off-target effects. In reproductive biology, CRISPR has advanced our understanding of fertility genes, allowed corrections of hereditary mutations, and opened avenues for novel therapeutic strategies. While its clinical application in human-assisted reproduction faces ethical and safety challenges, ongoing innovations hold promise for broader biomedical applications.

Recombinase polymerase amplification (RPA)-CRISPR/Cas12a assays have demonstrated remarkable potential for point-of-care detection of pathogens in resource-limited settings. Nevertheless, these assays fall short in delivering direct quantitative results due to the incompatibility between the RPA and CRISPR/Cas12a systems. To overcome this limitation, we developed a droplet pairing-merging enabled digital RPA-CRISPR/Cas12a (DIMERIC) assay in this study. By leveraging a microfluidic chip with a calabash-shaped microwell array, large-volume RPA droplets and small-volume CRISPR/Cas12a droplets were sequentially and size-selectively trapped, generating one-to-one droplet pairs. This spatial separation of the droplets eliminates the inhibitory effects of the CRISPR/Cas12a chemistry on RPA. Upon the completion of RPA, the CRISPR/Cas12a system can be activated by merging the paired droplets. This temporal separation of the RPA and CRISPR/Cas reactions allows for the accumulation of sufficient amplicons to efficiently unleash the collateral cleavage activity. The DIMERIC assay offers rapid quantification of nucleic acids, with the entire procedure being accomplished within 20 min. This assay was employed for the quantitative detection of Hepatitis B virus DNA from batched clinical serum samples, demonstrating a good correlation with qPCR (R2 = 0.92033) and ddPCR (R2 = 0.97337) outcomes. Consequently, the developed DIMERIC assay provides a valuable tool for rapid and precise quantification of pathogenic nucleic acids.

Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.

Rapeseed (Brassica napus L.) is a globally important oil crop, providing edible vegetable oil and other valuable sources for humans. Being an allotetraploid, rapeseed has a complex genome that has undergone whole-genome duplication, making molecular breeding rather difficult. Fortunately, clustered regularly interspacedshort palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies have emerged as a potent tool in plant breeding, providing unprecedented accuracy as well as effectiveness in genome editing. This review focuses on the application and progresses of CRISPR/Cas technologies in rapeseed. We discussed the principles and mechanisms of CRISPR/Cas systems focusing on their use in rapeseed improvement such as targeted gene knockout, gene editing and transcriptional regulation. Furthermore, we summarized the regulatory frameworks governing CRISPR-edited crops as well as the challenges and opportunities for their commercialization and adoption. The potential advantages of CRISPR-mediated traits in rapeseed such as increased yield, disease and stress resistance and oil quality are discussed along with biosafety and environmental implications. The purpose of this review is to provide insights into the transformative role of CRISPR/Cas technologies in rapeseed breeding and its potential to address global agricultural challenges while ensuring sustainable crop production.

Intrinsically disordered proteins (IDPs) and biomolecular condensates are critical for cellular processes and physiological functions. Abnormal biomolecular condensates can cause diseases such as cancer and neurodegenerative disorders. IDPs, including intrinsically disordered regions (IDRs), were previously considered undruggable due to their lack of stable binding pockets. However, recent evidence indicates that targeting them can influence cellular processes. This review explores current strategies to target IDPs and biomolecular condensates, potential improvements, and the challenges and opportunities in this evolving field.

CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.

For centuries, a competitive evolutionary race between prokaryotes and related phages or other mobile genetic elements has led to the diversification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‑associated sequence (Cas) genome‑editing systems. Among the different CRISPR/Cas systems, the CRISPR/Cas9 system has been widely studied for its precise DNA manipulation; however, due to certain limitations of direct DNA targeting, off‑target effects and delivery challenges, researchers are looking to perform transient knockdown of gene expression by targeting RNA. In this context, the more recently discovered type VI CRISPR/Cas13 system, a programmable single‑subunit RNA‑guided endonuclease system that has the capacity to target and edit any RNA sequence of interest, has emerged as a powerful platform to modulate gene expression outcomes. All the Cas13 effectors known so far possess two distinct ribonuclease activities. Pre‑CRISPR RNA processing is performed by one RNase activity, whereas the two higher eukaryotes and prokaryotes nucleotide‑binding domains provide the other RNase activity required for target RNA degradation. Recent innovative applications of the type VI CRISPR/Cas13 system in nucleic acid detection, viral interference, transcriptome engineering and RNA imaging hold great promise for disease management. This genome editing system can also be employed by the Specific High Sensitivity Enzymatic Reporter Unlocking platform to identify any tumor DNA. The discovery of this system has added a new dimension to targeting, tracking and editing circulating microRNA/RNA/DNA/cancer proteins for the management of cancer. However, there is still a lack of thorough understanding of the mechanisms underlying some of their functions. The present review summarizes the recent updates on the type VI CRISPR/Cas system in terms of its structural and mechanistic properties and some novel applications of this genome‑editing tool in cancer management. However, some issues, such as collateral degradation of bystander RNA, impose major limitations on its in vivo application. Furthermore, additional challenges and future prospects for this genome editing system are described in the present review.

Lactic acid bacteria (LABs) have a long history of use in food and beverages fermentation. Recently, several LABs have gained attention as starter or non-starter cultures and probiotics for making functional fermented foods, which have the potential to enhance human health. In addition, certain LABs show great potential as microbial cell factories for producing food-related chemicals. However, enhancing the outcomes of starter and non-starter cultures, exploring the complicated probiotic mechanism of LABs, and engineering strains to enhance the yields of high-value compounds for precision fermentation remains challenging due to the time-consuming and labor-intensive current genome editing tools. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system, originally an adaptive immune system in bacteria, has revolutionized genome editing, metabolic engineering and synthetic biology. Its versatility has resulted in extensive applications across diverse organisms. The widespread distribution of CRISPR-Cas systems and the diversity of CRISPR arrays in LAB genomes highlight their potential for studying the evolution of LABs. This review discusses the current advancement of CRISPR-Cas systems in engineering LABs for food application. Moreover, it outlines future research directions aimed at harnessing CRISPR-Cas systems to advance lactic acid bacterial research and drive innovation in food science.

Polymerase chain reaction (PCR), especially the multiplex PCR assay, enables simultaneous detection of multiple genes and is highly effective for diagnostic applications. The CRISPR-associated (Cas) system consists of several genes, and complete gene clusters are essential for its activity; multiplex PCR is an excellent method for detecting these multiple genes. This study focuses on the development and validation of a multiplex PCR protocol for the specific detection of CRISPR-Cas subtypes I-F1 and I-F2 found in A. baumannii, which is classified as a critical ESKAPE pathogen. The multiplex PCR method achieved a 100 % detection rate for isolates containing Cas subtypes I-F1 and I-F2 in clinical A. baumannii isolates. Testing across various genera and Acinetobacter species confirmed the high specificity of the assay, with no false positives, establishing it as a reliable tool for large-scale clinical applications. Of the 96 clinical A. baumannii isolates analysed, 29.167 % (n = 28) were multiplex PCR positive for a CRISPR-Cas system. Among these, 71.43 % (n = 20) had subtype I-F1, while 28.57 % (n = 8) had subtype I-F2. No clear association was found between Cas subtypes and resistance to the tested antibiotics or carbapenem genes. This study provides a valuable tool for monitoring CRISPR-Cas systems and can aid in various experimental and novel strategies to manage multidrug-resistant A. baumannii.

Clustered regularly interspaced short palindromic repeats (CRISPR) tools are revolutionizing the establishment of genotype-phenotype relationships and are transforming cell- and gene-based therapies. In the field of oncology, CRISPR/CRISPR-associated protein 9 (Cas9), Cas12, and Cas13 have advanced the generation of cancer models, the study of tumor evolution, the identification of target genes involved in cancer growth, and the discovery of genes involved in chemosensitivity and resistance. Moreover, preclinical therapeutic strategies employing CRISPR/Cas have emerged. These include the generation of chimeric antigen receptor T (CAR-T) cells and engineered immune cells, and the use of precision anticancer gene-editing agents to inactivate driver oncogenes, suppress tumor support genes, and cull cancer cells in response to genetic circuit output. This review summarizes the collective impact that CRISPR technology has had on basic and applied cancer research, and highlights the promises and challenges facing its clinical translation.

Cas proteins (CRISPR-associated protein) are the core components of the CRISPR-Cas system, playing critical roles in defending against foreign DNA and RNA invasions. Identifying Cas proteins can provide deeper insights into the immune mechanisms of the CRISPR-Cas system and help uncover the functional mechanisms of Cas proteins. In this study, we developed a computational tool named CasPro-ESM2, which combines the Pre-trained Protein Language Model ESM-2, multi-scale convolutional neural networks, and evolutionary information from protein sequences to identify Cas proteins. Experimental results demonstrate that CasPro-ESM2 outperforms existing models in Cas protein identification, achieving the highest values in metrics such as ACC, SP, SN, and MCC on two different datasets. Furthermore, we deployed this tool on a web server to enable direct access for users (http://www.bioai-lab.com/CasProESM-2).

CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates-containing a mismatch in the region complementary to RNA-by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a-crRNA complex and compared it with Cas9-sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a-crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9-sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.

The phosphodiester linkage is found in DNA, RNA and many signaling molecules, such as cyclic mononucleotide, cyclic dinucleotides (CDNs) and cyclic oligonucleotides (cONs). Enzymes that cleave the phosphodiester linkage (nucleases and phosphodiesterases) play important roles in cell persistence and fitness and have therefore become targets for various diseased states. While various inhibitors have been developed for nucleases and cyclic mononucleotide phosphodiesterases, and some have become clinical successes, there is a paucity of inhibitors of the recently discovered phosphodiesterases or ring nucleases that cleave CDNs and cONs. Inhibitors of bacterial c-di-GMP or c-di-AMP phosphodiesterases have the potential to be used as anti-virulence compounds, while compounds that inhibit the degradation of 3',3'-cGAMP, cA3, cA4, cA6 could serve as antibiotic adjuvants as the accumulation of these second messengers leads to bacterial abortive infection. In humans, 2'3'-cGAMP plays critical roles in antiviral and antitumor responses. ENPP1 (the 2'3'-cGAMP phosphodiesterase) or virally encoded cyclic dinucleotide phosphodiesterases, such as poxin, however, blunt this response. Inhibitors of ENPP1 or poxin-like enzymes have the potential to be used as anticancer and antiviral agents, respectively. This review summarizes efforts made towards the discovery and development of compounds that inhibit CDN phosphodiesterases and cON ring nucleases.

The miniature RNA-guided endonuclease IscB, as the evolutionary progenitor of Cas9, is attracting increased attention for genome editing due to its compact size and suitability for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells presents a significant challenge to its widespread application in precise site-specific human genome editing. In this study, we employed structure-guided rational design and protein engineering to optimize OgeuIscB, resulting in the identification of enIscB-F138R, which further enhanced editing activity up to 3.49-fold in mammalian cells compared to the high-activity OgeuIscB variant enIscB. Furthermore, we engineered an enIscB-F138R nickase-based adenine base editor, termed miABE-F138R, exhibiting enhanced base editing efficiency relative to miABE. To illustrate the practical applications of miABE-F138R, we applied it to rectify the prevalent R560C mutation in Pde6β associated with autosomal recessive retinitis pigmentosa, resulting in a significant improvement in activity compared to miABE. In conclusion, enIscB-F138R and miABE-F138R offer adaptable platforms for genome editing with potential significance in future biomedical applications.

This work comprehensively explores the effects of various positions of Cas12a split activators. Based on this, a multi-functional synergistic platform was constructed. The construction of a structural dynamic network, the sensitive detection of APE1, and a time-controlled photo-activation have been achieved, demonstrating the potential for expanding Cas12a applications.

Salmonella is considered as one of the primary pathogens associated with foodborne diseases globally. The effective treatment of these illnesses depends on the rapid and accurate identification of this organism. Traditional culture methods, however, necessitate extended testing periods, while many alternative techniques often lack precision. This research presents an innovative detection system that employs CRISPR-Cas12a for the detection of Salmonella. The detection system specifically targets the yfiR gene, which is amplified through isothermal exponential amplification (EXPAR). Target DNA hybridizes with the hairpin probe to form the DNA strand. The DNA strand was nicked to generate a nick by nicking endonuclease owing to its recognition sequence toward the hairpin probe. DNA polymerase can extend the 3'-end of the nicked site, which simultaneously displaces the newly synthesized strand. Thus, a large number of single-stranded DNA (ssDNA) were produced in the circle of nicking, polymerization, and strand displacement to achieve exponential amplification. The resultant amplified ssDNA products are subsequently recognized by CRISPR/Cas12a, resulting in the emission of a fluorescence signal. The detection system demonstrates a limit of detection of 10 fM for synthetic DNA and exhibits a strong linear relationship between 10 fM and 100 nM. Furthermore, the EXPAR-CRISPR/Cas12a detection system successfully identifies extracted genomic DNA samples containing Salmonella strains less than one hour, achieving a detection threshold of 1 pg/μL. This assay not only offers rapid results, requiring less than one hour for sample-to-answer outcomes, but is also cost-effective, minimizes aerosol risks, and provides exceptional specificity and sensitivity for the detection of Salmonella.

Recent advancements in synthetic biology and sequencing technologies have revolutionized the ability to manipulate viral genomes with unparalleled precision. This review focuses on two powerful methodologies: deep mutational scanning and CRISPR-based genome editing, that enable comprehensive mutagenesis and detailed functional characterization of viral proteins. These approaches have significantly deepened our understanding of the molecular determinants driving viral evolution and adaptation. Furthermore, we discuss how these advances provide transformative insights for future vaccine development and therapeutic strategies.

Insertion sequences (ISs) are key components of most bacterial genomes and play a crucial role in bacterial mutagenesis. In this study, we observed the insertion of an IS element, ISLrh, from the Lacticaseibacillus rhamnosus M1 genome into plasmid pGK12, resulting in the generation of a new plasmid, pTRK829. This insertion enabled pTRK829 to replicate in hosts previously incompatible with pGK12, including L. rhamnosus M1, L. rhamnosus GG (LGG), Lacticaseibacillus casei ATCC 393, and Lacticaseibacillus paracasei ATCC 25598. However, the ISLrh-inserted plasmid, pTRK829, was unstable and underwent a spontaneous deletion, resulting in a smaller and more stable variant, pTRK830, which retained ISLrh. Characterization of pTRK829 and pTRK830 across several host strains showed that ISLrh insertion led to a dramatic alteration in host range and impacted plasmid stability and copy number. Sequence and functional analysis of pTRK830 revealed that the terminal inverted repeats (IRs) of the inserted ISLrh and its insertion location were essential for plasmid replication in LGG. Finally, pTRK830 was successfully used as an expression vector for heterologous β-glucuronidase expression in LGG, L. casei ATCC 393, and L. paracasei ATCC 25598. In conclusion, this study demonstrated that the insertion of the IRs from ISLrh at a specific location can directly change the host range and stability of pGK12. Furthermore, we also demonstrated the potential of pTRK830 as a new cloning and expression vector for genetically intractable lactobacilli.

This study highlights the significant impact of insertion sequence (IS) elements on plasmid replication in lactobacilli. The spontaneous integration of an IS element from the Laticaseibacillus rhamnosus genome into plasmid pGK12 not only expands its host range in previously incompatible strains but also changes plasmid stability and copy number. This expansion of the plasmid's host range is crucial for developing versatile genetic tools across diverse lactobacilli species. Additionally, the stable plasmid variant of pGK12 with the IS element insertion offers a valuable tool for cloning and gene expression in lactobacilli. These findings enhance our understanding of plasmid-IS element interactions and may provide insight into a new method to expand the host range of existing plasmids.

As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are wildly studied for their unique asymmetric life cycle and versatile applications in industry, agriculture, and biomedicine. However, genetic manipulation in these bacteria remains challenging, typically requiring time-consuming and labor-intensive procedures. Here, we report a practical CRISPR-SpCas9M-reporting system that overcomes the limitations of SpCas9 expression and CRISPR escape, enabling efficient, markerless, and rapid genome editing in C. crescentus. Two genes encoding for a pair of scaffold proteins were knocked out individually or iteratively, demonstrating their direct involvements in cellular signaling asymmetry. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized in the system, finally achieving the apparent editing efficiency up to 80% in C. crescentus. Furthermore, we applied the CRISPR-SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and general editing strategy. We anticipate that this system could be applied to other CRISPR-Cas-recalcitrant organisms, accelerating both basic and applied research in α-proteobacteria.

With the widespread application of the CRISPR-Cas system in gene editing and related fields, along with the increasing availability of metagenomic data, the demand for detecting and classifying CRISPR-Cas systems in metagenomic data sets has grown significantly. Traditional classification methods for CRISPR-Cas systems primarily rely on identifying cas genes near CRISPR arrays. However, in cases where cas gene information is absent, such as in metagenomes or fragmented genome assemblies, traditional methods may fail. Here, we present a deep learning-based method, CRISPRclassify-CNN-Att, which classifies CRISPR loci solely based on repeat sequences. CRISPRclassify-CNN-Att utilizes convolutional neural networks (CNNs) and self-attention mechanisms to extract features from repeat sequences. It employs a stacking strategy to address the imbalance of samples across different subtypes and uses transfer learning to improve classification accuracy for subtypes with fewer samples. CRISPRclassify-CNN-Att demonstrates outstanding performance in classifying multiple subtypes, particularly those with larger sample sizes. Although CRISPR loci classification traditionally depends on cas genes, CRISPRclassify-CNN-Att offers a novel approach that serves as a significant complement to cas-based methods, enabling the classification of orphan or distant CRISPR loci. The proposed tool is freely accessible via https://github.com/Xingyu-Liao/CRISPRclassify-CNN-Att.

The increasing feasibility of whole-genome sequencing has been highly anticipated, promising to transform our understanding of the biology of nonmodel species. Notably, dramatic cost reductions beginning around 2007 with the advent of high-throughput sequencing inspired publications heralding the 'genomics revolution', with predictions about its future impacts. Although such predictions served as useful guideposts, value is added when statements are evaluated with the benefit of hindsight. Here, we review 10 key predictions made early in the genomics revolution, highlighting those realised while identifying challenges limiting others. We focus on predictions concerning applied aspects of genomics and examples involving salmonid species which, due to their socioeconomic and ecological significance, have been frontrunners in applications of genomics in nonmodel species. Predicted outcomes included enhanced analytical power, deeper insights into the genetic basis of phenotype and fitness variation, disease management and breeding program advancements. Although many predictions have materialised, several expectations remain unmet due to technological, analytical and knowledge barriers. Additionally, largely unforeseen advancements, including the identification and management applicability of large-effect loci, close-kin mark-recapture, environmental DNA and gene editing have added under-anticipated value. Finally, emerging innovations in artificial intelligence and bioinformatics offer promising new directions. This retrospective evaluation of the impacts of the genomic revolution offers insights into the future of genomics for nonmodel species.

The energetic basis for the enhanced PAM (protospacer adjacent motif) readability in engineered Cas9-NG (a variant of Cas9 from Streptococcus pyogenes (SpCas9)) with seven mutations: (R1335V, E1219F, D1135V, L1111R, T1337R, G1218R, and A1322R) remains a fundamental unsolved problem. Utilizing the X-ray structure of the precatalytic complex (SpCas9:sgRNA:dsDNA) as a template, we calculated the changes in PAM (TGG, TGA, TGT, or TGC) binding affinity (ΔΔG) associated with each of the seven mutations in SpCas9 through rigorous alchemical simulations (sampling ∼ 53 μs). The underlying thermodynamics (ΔΔG) accounts for the experimentally observed differences in DNA cleavage activity between SpCas9 and Cas9-NG across various DNA substrates. The interaction energies between SpCas9 and DNA are significantly influenced by the type and location of the amino acid mutations. Notably, the R1335V mutation disfavors DNA binding by disrupting critical interactions with the PAM. However, the destabilizing effect of the R1335V mutation is mitigated by four advantageous mutations (E1219F, D1135V, L1111R, and T1337R), which primarily introduce nonbase-specific interactions and enhance PAM readability. The hydrophobic substitutions (E1219F and D1135V) are particularly impactful, as they exclude solvent from the PAM binding pocket, strengthening electrostatic interactions in the low dielectric medium and increasing the stability of the noncognate PAM complexes by ∼2-5 kcal/mol. Additionally, L1111R and T1337R facilitate DNA binding by forming direct electrostatic contacts. In contrast, the charge mutations G1218R and A1322R do not effectively promote interactions with the negatively charged DNA, clearly demonstrating that the location of mutations is crucial in shaping these interaction energetics. We demonstrated that stabilization of the Cas9-NG: noncognate PAM complexes enables broader PAM recognition. This is primarily achieved through two mechanisms: (1) the establishment of new nonbase-specific interactions between the protein and nucleotides and (2) the enhancement of electrostatic interactions within a relatively dry and hydrophobic pocket. The findings revealed that mutation-induced desolvation can improve the recognition of noncognate PAMs, paving the way for the rational and innovative design of SpCas9 mutants.

Type III CRISPR-Cas systems defend against viral infection in prokaryotes using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligo-adenylate (cOA) messengers to activate CARF immune effectors. Here we investigated a protein containing a CARF domain fused Toll/interleukin-1 receptor (TIR) domain, Cat1. We found that Cat1 provides immunity by cleaving and depleting NAD+ molecules from the infected host, inducing a growth arrest that prevents viral propagation. Cat1 forms dimers that stack upon each other to generate long filaments that are maintained by bound cOA ligands, with stacked TIR domains forming the NAD+ cleavage catalytic sites. Further, Cat1 filaments assemble into unique trigonal and pentagonal networks that enhance NAD+ degradation. Cat1 presents an unprecedented chemistry and higher-order protein assembly for the CRISPR-Cas response.

Type III-E CRISPR-Cas effectors, referred to as Cas7-11 or giant Repeat-Associated Mysterious Protein, are single proteins that cleave target RNAs (tgRNAs) without nonspecific collateral cleavage, opening new possibilities for RNA editing. Here, biochemical assays combined with amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments reveal the dynamics of apo Cas7-11. The HDX-MS results suggest a mechanism by which CRISPR RNA (crRNA) stabilizes the folded state of the protein and subsequent tgRNA binding remodels it to the active form. HDX-MS shows that the four Cas7 RNA recognition motif (RRM) folds are well-folded, but insertion sequences, including disordered catalytic loops and β-hairpins of the Cas7.2/Cas7.3 active sites, fold upon binding crRNA leading to stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. TgRNA binding causes conformational changes around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot independently process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces tgRNA cleavage. The insertion domain interacts most stably with mature crRNA. Finally, we show a crRNA-induced conformational change in one of the tetratricopeptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding.

The discovery and subsequent characterization and applications of CRISPR-Cas is one of the most fascinating scientific stories from the past two decades. While first identified in Escherichia coli, this microbial workhorse often took a back seat to other bacteria during the early race to detail CRISPR-Cas function as an adaptive immune system. This was not a deliberate slight, but the result of early observations that the CRISPR-Cas systems found in E. coli were not robust phage defense systems as first described in Streptococcus thermophilus. This apparent lack of activity was discovered to result from transcriptional repression by the nucleoid protein H-NS. Despite extensive evidence arguing against such roles, some studies still present E. coli CRISPR-Cas systems in the context of anti-phage and/or anti-plasmid activities. Here, the studies that led to our understanding of its cryptic nature are highlighted, along with ongoing research to uncover potential alternative functions in E. coli.

Oral potentially malignant disorders (OPMDs) and gastrointestinal precancerous lesions (GPLs) are major public health concerns because of their potential to progress to cancer. Probiotics, prebiotics, and engineered probiotics can positively influence the prevention and management of OPMDs and GPLs. This review aims to comprehensively review the application status of probiotics, prebiotics and engineered probiotics in OPMDs and GPLs, explore their potential mechanisms of action, and anticipate their future clinical use.

The CRISPR-Cas system is a well-known adaptive immune system in bacteria, and a prominent mechanism for evading this immunity involves anti-CRISPR (Acr) proteins, which employ various methods to neutralize the CRISPR-Cas system. In this study, using structural and biochemical analyses, we revealed that AcrIE7 binds to the single-stranded DNA in the R-loop formed when Cascade encounters the target DNA, thereby preventing Cas3 from cleaving the DNA. This represents a different inhibition strategy distinct from previously reported Acr mechanisms and offers insights into CRISPR-Cas inhibition.

CRISPR/Cas9 technology has revolutionized genetic and biomedical research in recent years. It enables editing and modulation of gene function with an unparalleled precision and effectiveness. Among the various applications and prospects of this technology, the opportunities it offers in unraveling the molecular underpinnings of a myriad of central nervous system diseases, including neurodegenerative disorders, psychiatric conditions, and developmental abnormalities, are unprecedented. In this review, we highlight the applications of CRISPR/Cas9-based therapeutics as a promising strategy for management of Alzheimer's disease and transformative impact of this technology on AD research. Further, we emphasize the role of CRISPR/Cas9 in generating accurate AD models for identification of novel therapeutic targets, besides the role of CRISPR-based therapies aimed at correcting AD-associated mutations and modulating the neurodegenerative processes. Furthermore, various delivery systems are reviewed and potential of the non-viral nanotechnology-based carriers for overcoming the critical limitations of effective delivery systems for CRISPR/Cas9 is discussed. Overall, this review highlights the promise and prospects of CRISPR/Cas9 technology for unraveling the intricate molecular processes underlying the development of AD, discusses its limitations, ethical concerns and several challenges including efficient delivery across the BBB, ensuring specificity, avoiding off-target effects. This article can be helpful in better understanding the applications of CRISPR/Cas9 based therapeutic approaches and the way forward utilizing enormous potential of this technology in targeted, gene-specific treatments that could change the trajectory of this debilitating and incurable illness.

The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans provides defense against mobile genetic elements in the absence of target DNA degradation. In recent studies, Escherichia coli BL21-AI cells with Type IV-A1 CRISPR-Cas activity displayed a heterogeneous colony growth phenotype. Here, we developed a convenient smartphone-mediated automatic remote-controlled time-lapse imaging system (SMARTIS), that enables monitoring of growing bacteria over time. The system's design includes a custom-built imaging box equipped with LED lights, an adjustable heating system and a smartphone that can be remotely controlled using freely available, user-friendly applications. SMARTIS allowed long-term observation of growing colonies and was utilized to analyze different growth behaviors of E. coli cells expressing Type IV-A1 CRISPR ribonucleoproteins. Our findings reveal that heterogeneity in colonies can emerge within hours of initial growth. We further examined the influence of different expression systems on bacterial growth and CRISPR interference activity and demonstrated that the observed heterogeneity of colony-forming units is strongly influenced by plasmid design and backbone identity. This study highlights the importance of careful assessment of heterogenous colony growth dynamics and describes a real-time imaging system with wide applications beyond the study of CRISPR-Cas activity in bacterial hosts.

Multi-drug resistant and carbapenem-resistant hypervirulent Klebsiella pneumoniae strains are spreading globally at an alarming rate, emerging as one of the most serious threats to global public health. The formidable challenges posed by the current arsenal of antimicrobials highlight the urgent need for novel strategies to combat K. pneumoniae infections. This review begins with a comprehensive analysis of the global dissemination of virulence factors and critical resistance profiles in K. pneumoniae, followed by an evaluation of the accessibility of novel therapeutic approaches for treating K. pneumoniae in clinical settings. Among these, phage therapy stands out for its considerable potential in addressing life-threatening K. pneumoniae infections. We critically examine the existing preclinical and clinical evidence supporting phage therapy, identifying key limitations that impede its broader clinical adoption. Additionally, we rigorously explore the role of genetic engineering in expanding the host range of K. pneumoniae phages, and discuss the future trajectory of this technology. In light of the 'Bad Bugs, No Drugs' era, we advocate leveraging artificial intelligence and deep learning to optimize and expand the application of phage therapy, representing a crucial advancement in the fight against the escalating threat of K. pneumoniae infections.

Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.

Social bees, with their specialized gut microbiota and societal transmission between individuals, provide an ideal model for studying host-gut microbiota interactions. While the functional disparities arising from strain-level diversity of gut symbionts and their effects on host health have been studied in Apis mellifera and bumblebees, studies focusing on host-specific investigations of individual strains across different honeybee hosts remain relatively unexplored. In this study, the complete genomic sequences of 17 strains of Gilliamella from A. mellifera, Apis cerana and Bombus terrestris were analyzed. The analysis revealed that the strains of A. mellifera display a more expansive genomic and functional content compared to the strains of A. cerana and B. terrestris. Phylogenetic analysis showed a deep divergence among the Gilliamella strains from different hosts. Additionally, biochemistry tests and antibiotic susceptibility tests revealed that gut strains from A. mellifera exhibited a more extensive pathway for carbohydrate metabolism and a greater resistance to antibiotics than gut strains from A. cerana and B. terrestris. Strains from A. mellifera and A. cerana showed higher colonization efficiency and competitive ability whithin their respective host species, indicating a higher degree of host-specific adaptation of local gut microbiota. In addition, colonization by A. mellifera-derived strain triggers a stronger transcriptional response in the host than A. cerana-derived strain. The variation in the number of differentially expressed genes and the involvement of distinct signaling pathways across these two host species suggest species-specific adaptations to Gilliamella strains. These findings suggest that despite occupying similar niches in the bee gut, strain-level variations can influence microbial functions, and their impact on host physiological functions may vary across different strains.

Organoids represent a significant advancement in disease modeling, demonstrated by their capacity to mimic the physiological/pathological structure and functional characteristics of the native tissue. Recently CRISPR/Cas9 technology has emerged as a powerful tool in combination with organoids for the development of novel therapies in preclinical settings. This review explores the current literature on applications of pooled CRISPR screening in organoids and the emerging role of these models in understanding cancer. We highlight the evolution of genome-wide CRISPR gRNA library screens in organoids, noting their increasing adoption in the field over the past decade. Noteworthy studies utilizing these screens to investigate oncogenic vulnerabilities and developmental pathways in various organoid systems are discussed. Despite the promise organoids hold, challenges such as standardization, reproducibility, and the complexity of data interpretation remain. The review also addresses the ideas of assessing tumor organoids (tumoroids) against established cancer hallmarks and the potential of studying intercellular cooperation within these models. Ultimately, we propose that organoids, particularly when personalized for patient-specific applications, could revolutionize drug screening and therapeutic approaches, minimizing the reliance on traditional animal models and enhancing the precision of clinical interventions.

CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.

Epithelial ovarian carcinoma (EOC), the most lethal gynecologic cancer, is often diagnosed at advanced stages, which urge us to explore the novel therapeutic strategies. Mouse models have played a crucial role in elucidating the molecular mechanisms for the development ovarian cancer and its therapeutic strategies. However, there are still various challenges in modeling the genetic drivers of ovarian cancer in animal models. Here, we provided an overview of the research advances for the molecular mechanisms underlying EOC development, therapeutic strategies, the CRISPR genome editing technology and its generated EOC models. The review also comprehensively discussed the advantages and obstacles of CRISPR in generating EOC mouse models and the promising therapeutic approach by correcting the oncogenes of EOC through in vivo delivery of gene-edited components. The development of more precise animal models, along with a deeper understanding of EOC molecular mechanisms, will dramatically benefit the investigation and treatment of EOC.

Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in Nicotiana benthamiana using the CRISPR-Cas12a multiplex gene editing system. We employed two effector proteins LbCas12a and FnCas12a coupled with six guide RNAs targeting virus genome and a novel mRNA-gRNA nucleic acid complex to transport gRNA efficiently. Compared with the BSCTV accumulation in the wild-type N. benthamiana, it was reduced by more than 90% by most transgenic events derived at 7 days post-inoculation. Additionally, the shoot-tip leaves were normal in the transgenic plants, whereas they appeared severely curled and stunted in wild-type N. benthamiana at 15 days post-infection. Target sites evaluation revealed that the editing system can directly destroy the structure of BSCTV viral genomes via large fragment deletions. We quantified TMV virus accumulation in the transgenic N. benthamiana lines by monitoring dynamic changes in GFP fluorescence and quantitative analysis by qPCR showed that the CRISPR-Cas12a system can introduce TMV virus resistance to N. benthamiana by preventing its systemic spread. Our study provides an innovative strategy-an mRNA-gRNA nucleic acid complex-which has proven to be highly effective in the gene-editing system and offers an efficient antiviral approach for generating virus-resistant plants.

Several Cas12a variants have been developed to broaden its targeting range, improve the gene editing specificity or the efficiency. However, selecting the appropriate Cas12a among the many orthologs for a given target sequence remains difficult. Here, we perform high-throughput analyses to evaluate the activity and compatibility with specific PAMs of 24 Cas12a variants and develop deep learning models for these Cas12a variants to predict gene editing activities at target sequences of interest. Furthermore, we reveal and enhance the truncation in the integrated tag sequence that may hinder off-targeting detection for Cas12a by GUIDE-seq. This enhanced system, which we term enGUIDE-seq, is used to evaluate and compare the off-targeting and translocations of these Cas12a variants.

CRISPR system has been widely used due to its precision and versatility in gene editing. Un1Cas12f1 from uncultured archaeon (hereafter referred to as Cas12f), known for its compact size (529 aa), exhibits obvious delivery advantage for gene editing in vitro and in vivo. However, its activity remains suboptimal. In this study, we engineer circular guide RNA (cgRNA) for Cas12f and significantly improve the efficiency of gene activation about 1.9-19.2-fold. When combined with a phase separation system, the activation efficiency is further increased about 2.3-3.9-fold. In addition, cgRNA enhances the editing efficiency and narrows the editing window of adenine base editing about 1.2-2.5-fold. Importantly, this optimization strategy also boosts the Cas12f-induced gene activation efficiency in mouse liver. Therefore, we demonstrate that cgRNA is able to enhance Cas12f-based gene activation and adenine base editing, which holds great potential for gene therapy.

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis, after which it remains catalytically active and non-specifically cleaves ssDNA in trans. Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied 'NUC loop', integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.

Chromatin organization is a relevant layer of control of gene expression during plant development. Chromatin states strictly depend on associated features such as DNA methylation, histone modifications and histone variants. Thus, epigenome editing has become of primary interest to alter gene expression without disrupting genomic sequences. Different tools have been developed to address this challenge, starting with modular Zinc Finger Proteins (ZFPs) and Transcription Activator Like Effectors (TALEs). However, the discovery of CRISPR/Cas9 system and the adaptability of technologies based on enzymatically dead Cas9 (dCas9) have paved the way towards a reliable and adaptable epigenome editing in a great variety of organisms. In this review, we will focus on the application of targeted epigenome editing technologies in plants, summarizing the most updated advances in this field. The promising results obtained by altering the expression state of targets involved in flowering time and abiotic stress resistance are crucial not only for elucidating the molecular interactions that underly chromatin dynamics, but also for future applications in breeding programs as an alternative route to genetic manipulation towards the achievement of higher quality crops particularly in terms of nutritional properties, yield and tolerance.

For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes, remain significantly less accessible. Here, we describe the production, delivery, and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors.