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The Codon Usage Bias Analysis of Free-Living Ciliates' Macronuclear Genomes and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Vector Construction of Stylonychia lemnae.
Ciliates represent higher unicellular animals, and several species are also important model organisms for molecular biology research. Analyses of codon usage bias (CUB) of the macronuclear (MAC) genome in ciliates can not only promote a better understanding of the genetic mode and evolution history of these organisms but also help optimize codons to improve the gene editing efficiency of model ciliates. In this study, macronuclear genome sequences of nine free-living ciliates were analyzed with CodonW software to calculate the following indices: the guanine-cytosine content (GC); the frequency of the nucleotides U, C, A, and G at the third position of codons (U3s, C3s, A3s, G3s); the effective number of codons (ENC); the correlation between GC at the first and second positions (GC12); the frequency of the nucleotides G + C at the third position of synonymous codons (GC3s); the relative synonymous codon usage (RSCU). Parity rule 2 plot analysis, neutrality plot analysis, and correlation analysis were performed to explore the factors that influence codon preference. The results showed that the GC contents in nine ciliates' MAC genomes were lower than 50% and appeared AT-rich. The base compositions of GC12 and GC3s are markedly distinct and the codon usage pattern and evolution of ciliates are affected by genetic mutation and natural selection. According to the synonymous codon analysis, the codons of most ciliates ended with A or U and eight codons were the general optimal codons of nine ciliates. A clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) expression vector of Stylonychia lemnae was constructed by optimizing the macronuclear genome codon and was successfully used to knock out the Adss gene. This is the first such extensive investigation of the MAC genome CUB of ciliates and the initial successful application of the CRISPR/Cas9 technique in free-living ciliates.
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Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon.
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Development of a versatile nuclease prime editor with upgraded precision.
The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential.
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Systematic synthesis of CRISPR/Cas applications for enhancing salt tolerance in crops: a decade of progress and challenges.
Soil salinity is a major constraint on global crop productivity, driving the need for salt-tolerant varieties. While CRISPR-Cas genome editing offers targeted solutions for trait improvement, significant biological and technical bottlenecks limit its application in conferring salt stress resilience. This systematic summarizes findings from 83 peer-reviewed studies (2015-2024) employing CRISPR/Cas technologies to improve salt tolerance in five major crops (rice, wheat, maize, sorghum, barley). Our systematic review reveals that early single-gene edits achieved modest gains (30-50% Na⁺ exclusion) but often showed limited yield gains in field settings, potentially due to compensatory regulation and environmental variation. The literature suggests that multiplex designs spanning ion homeostasis, osmoprotection, and ROS management can improve salt-tolerance outcomes and help maintain yield under severe salinity; however, the magnitude of benefit varies with crop, genotype, and transformation/regeneration context. Protein-protein interaction networks identified 12 hub genes and three functional modules, highlighting SOS3 and MPK6 as critical bottlenecks whose disruption risks pleiotropic effects. Spatial expression analysis underscored tissue-specific trade-offs, constitutive editing of root-dominant genes in shoots reduced yields by 15-28%, while tissue-optimized promoters minimized physiological conflicts. Persistent challenges include genotype-dependent transformation inefficiencies, epigenetic drift and environmental interactions under salt stress. Collectively, our synthesis consolidates and refines current best practices for salt-tolerance genome editing and highlights major bottlenecks-particularly regeneration/transformability, genotype dependence, and epigenetic constraints-that should be explicitly considered in experimental design and reporting.
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Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.
Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.
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One-Step Biological Upcycling of Chitin Wastes into Violacein by Engineered Chromobacterium violaceum.
Existing technologies for the valorization of organic wastes have been focused mainly on degradable wastes, while an efficient, low-carbon approach for the upcycling of shell waste is still lacking. Here, we report a one-step chitin biological fermentation process (CBFP), based on the construction of Chromobacterium violaceum engineered strain, for efficiently converting shell waste-derived chitin into high-value violacein. A high-efficiency CRISPR cytosine-base editor (pRK2-BE, 97% editing efficiency) was developed for C. violaceum, which demonstrated cv_4240, cv_1440, and cv_2935 as the major chitin hydrolysis genes and phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) as the major N-acetyl-glucosamine uptake pathway. The engineered strain WT/pBAD-4, co-overexpressing of cv_4240, cv_1440, cv_2935, and vioABCDE, efficiently utilized colloidal chitin and crystalline chitin as the sole carbon and nitrogen source, achieving violacein yields of 159.78 and 120.95 mg·L-1, respectively. This study provided an economically viable and environmentally sustainable solution for green upcycling of shell waste.
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Are the current gRNA ranking prediction algorithms useful for genome editing in plants?
Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.
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CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction.
Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards L-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an L-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces L-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Genome Editing in Inherited Cardiovascular Diseases: Therapeutic Advances, Emerging Editing Platforms, and Translational Challenges.
Inherited cardiovascular diseases (ICVDs) are a broad category of hereditary disorders resulting from pathogenic mutations that alter cardiac anatomy, electrical conduction, and metabolic pathways. The main types include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), cardiac channelopathies, and familial hypercholesterolemia (FH) and are associated with heart failure, malignant arrhythmias, and sudden cardiac death, particularly in young people. Current treatments, such as pharmacological therapies, implantable cardioverter-defibrillators, and heart transplantation, have significantly improved outcomes; however, these treatments are mainly targeted toward symptom control and the prevention of complications without correcting the underlying genetic defects. Recent advancements in genome editing have transformed precision cardiovascular care, with clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies emerging as potential methods for targeting and modifying disease-causing variants. Emerging platforms, such as base editing, prime editing, and epigenome editing, have widened the therapeutic potential of genome engineering by enhancing editing precision and reducing some of the constraints of the traditional CRISPR-Cas9 strategy. In the setting of inherited cardiovascular disorders, promising early results have been obtained in preclinical studies using human induced pluripotent stem cell and animal models, including correction of MYBPC3 and MYH7 mutations in HCM; targeting of TTN-, RBM20-, and LMNA-associated mechanisms in DCM; restoration of ion channel function in inherited arrhythmia syndromes; and modulation of lipid-related genes such as PCSK9, LDLR, and ANGPTL3 in FH. However, major challenges remain, including off-target effects, effective delivery to cardiac tissues, immunological responses, interpretation of genetic variants, and ethical concerns. This review provides a translational, integrated view of the evolution of CRISPR-based genome editing technologies, their application to ICVDs, current preclinical and clinical advances, and the challenges that need to be addressed to achieve safe and effective translation into cardiovascular therapeutics.
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CRISPR-based one-pot detection: A game-changer in nucleic acid analysis.
The CRISPR/Cas system, originally developed as a gene-editing tool, has rapidly emerged as a powerful platform for nucleic acid detection due to its remarkable specificity, programmability, and robust trans-cleavage activity. While conventional CRISPR-based assays typically require separate amplification and detection steps, introducing complexity, manual intervention, and contamination risks. Recent innovations have led to the development of one-pot detection strategies that integrate target amplification with Cas-mediated signal generation within a single reaction vessel. These streamlined systems not only reduce handling steps and turnaround time but also enhance sensitivity and enable multiplexed detection of low-abundance nucleic acid targets. As such, one-pot CRISPR diagnostics are poised to transform point-of-care testing, pathogen surveillance, and clinical diagnostics. In this review, we summarize the latest advances in one-pot CRISPR-based detection technologies, evaluate their current limitations, and discuss strategies for optimization. Future efforts should focus on refining reaction kinetics, improving reagent stability, reducing costs, and expanding the repertoire of detectable targets. Furthermore, integration with portable platforms, AI-assisted signal interpretation, and the discovery of novel Cas effectors will be pivotal in realizing the full potential of CRISPR-based one-pot systems in diverse real-world applications.
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