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PubMed2026

Heavy metal stress in native plant species: investigating phytoremediation potential through physiological and ISSR/SCoT molecular assessments.

In emerging countries, increased industrial activity has a significant impact on economic growth and urban development. However, the acceleration of industrial processes is accompanied by the release of contaminants such as heavy metals. According to the World Health Organization, one-fourth of all human diseases are caused by environmental contaminants, including heavy metals, which can impair numerous organs such as the neurological system, liver, and reproductive systems. This increased efforts to find effective and sustainable methods to remove heavy metals. Phytoremediation is an environmentally benign method of removing heavy metals using specific plants. Thus, from industrially contaminated locations, common native plant species of Lactuca serriola, Sisymbrium irio, Chenopodium murale, and Cynanchum acutum were selected for this study to assess the mechanisms of their molecular and physiological tolerance. Soil and plants were tested for heavy metals (Cd, Pb, and Cu), and contaminated locations were classified as low and highly polluted. Measurements were made of soluble sugar, protein, secondary metabolites, malondialdehyde, and H2O2. Additionally, inter simple sequence repeat (ISSR), start codon targeted (SCoT), and genomic template stability GTS were used. In heavily polluted areas, all plant species exhibit elevated amounts of sugar, proteins, H2O2, MDA, and secondary metabolites, while total phenolics showed a unique significant interaction (plant-location), where Cynanchum exhibited a hyper-stress phenolic accumulation to cope with toxicity, whereas Chenopodium maintained genomic stability with balanced phenolic level. Based on these findings, both Cynanchum acutum and Chenopodium murale demonstrate superior potential for phytoremediation and warrant further investigation for ecological restoration.

Heavy metal↗
PubMed2026

EST-SSR based genetic polymorphism among Lablab (Lablab purpureus L. Sweet) accessions contrasting for drought stress at seedling stage.

Lablab is a multipurpose and the most drought-tolerant (DT) crop compared with its relatives. Despite its potential, Lablab is still an underutilized crop with a lack of improved varieties in many countries. The DT (D349, D147, HA4, D363, D352, D359, D348, D311, D55 and D250) and drought-susceptible (DS) (D271, D66, D106, D6, D26, D255, D28, D186, D95, and D258) accessions were earlier identified according to their morphological and biochemical responses to moisture stress at the seedling stage. These accessions were used to establish genetic polymorphism among the accessions contrasting for drought stress based on the Expressed Sequence Tag-Simple Sequence Repeats (EST-SSR) markers. The CTAB protocol was employed for the genomic DNA extraction. After DNA quality and quantity verification, the PCR was conducted using 16 EST-SSR primer pairs specific to the Lablab. The products were separated through the horizontal polyacrylamide gel electrophoresis (hPAGE). Discriminating ability of the markers and primers' efficiency were evaluated based on various genetic parameters. Principal Coordinate Analysis (PCoA) was performed to estimate the distance matrix among the population and among the accessions. While cluster analysis was processed to trace the genetic relationship among the accessions, dendrogram was constructed to decipher their genetic relationship. Analysis of Molecular Variance (AMOVA) was finally computed to quantify the diversity level and genetic relationship among the population, and among the accessions. A low polymorphism (GD = 0.19) was observed between the DT and DS accessions, likely due to limited discriminatory power of the EST-SSR markers. However, the PCoA, cluster analysis and AMOVA identified DT (D147, HA4, and D349) and DS (D106, D95, and D271) accessions as strongly contrasting populations under drought stress, with D147, HA4, D349, D363, D359, D352, and D348 further recommended as DT accessions. Given the low polymorphism observed, further validation using more informative molecular markers and advanced genomic approaches is recommended to improve the identification of drought-tolerance genes and related QTLs to support Lablab breeding programs.

Expressed Sequence Tags↗
PubMed2026

Molecular investigation of the progenitors, origin and domestication patterns of diploid Chinese old garden roses.

BACKGROUND AND AIMS: Chinese old garden roses are major contributors to the genetic development of modern roses. The RoKSN gene is associated with continuous flowering in roses and is proposed to have originated from Chinese wild roses. However, the wild roses that are implicated in the breeding of Chinese old garden roses and the origin of the RoKSN locus remain unidentified. We collected 25 of the most renowned and classic diploid Chinese old garden roses along with all related wild roses from East Asia. These roses were analysed with the aim of identifying the wild species that contributed to the genetic composition of Chinese old garden roses. In addition, we aimed to infer the geographical origin of the RoKSN gene and to develop a schematic overview of hybrid domestication of Chinese old garden roses. METHODS: We compared the haplotypes of internal transcribed spacers (nrITS), six nuclear single-copy genes and three chloroplast genes between Chinese old garden roses and wild roses. Additionally, we assessed genetic organization using 21 expressed sequence tag-simple sequence repeats to identify potential donor species that contributed to the emergence of these cultivars. Primers were designed for RoKSN to allow comparison of the gene across the entire distribution range of Rosa sect. Chinenses. KEY RESULTS: Our findings confirmed that the majority of rose cultivars are descendants of early hybridization events. Rosa chinensis var. spontanea, R. odorata var. gigantea and R. multiflora var. cathayensis were the primary donors for the 25 cultivar roses. Chinese old garden roses were categorized into four groups. Ten cultivars were hybrids between R. chinensis var. spontanea and R. multiflora var. cathayensis, thereby forming the 'Old Blush' group. Five cultivars were hybrids between 'Old Blush' and the R. kwangtungensis species complex, thereby forming the 'Slater's crimson' group. Six cultivars were hybrids between 'Old Blush' and R. odorata var. gigantea, thereby forming the 'Tea Rose' group, and three cultivars were hybrids that evolved from more than three donors. Moreover, we observed relatively close genetic proximity among Chinese old garden roses with an identical RoKSN-copia gene that is responsible for continuous flowering, which indicates a single origin for this retrotransposon-containing allele. Additionally, we determined that the haplotypes of the RoKSN-copia gene predominantly occurred in the Sichuan Basin region. In contrast, R. chinensis cultivated in the Ya'an region showed no markers of hybridization and displayed a genetic composition that was close to that of the wild species R. chinensis var. spontanea. This cultivar may represent the earliest mutated individual that bears the RoKSN-copia gene and may have served as a bridge from wild species to continuous-flowering old rose cultivars. CONCLUSIONS: The study provides crucial evidence that elucidates the origin of cultivated roses and lays the groundwork for further analysis of the breeding history of Chinese old garden roses using genomic data.

Domestication↗
PubMed2025

Genomic signature and evolutionary history of completely cleistogamous lineages in the non-photosynthetic orchid Gastrodia.

Despite a long-standing interest since Darwin's time, the genomic implications of obligate self-fertilization remain elusive. Complete cleistogamy-the obligate production of closed, self-pollinating flowers-represents an extreme reproductive strategy. Here, we present the genomic profiles and evolutionary history of two lineages of the mycoheterotrophic orchid Gastrodia, both of which independently acquired complete cleistogamy, based on detailed sampling and a combination of simple sequence repeat (SSR), multiplexed ISSR genotyping by sequencing (MIG-seq) and RNA-seq data. Our analysis reveals clear species delimitation, with no evidence of introgression between the completely cleistogamous species and their co-occurring allogamous sisters. Intriguingly, all analyses indicate that both the completely cleistogamous Gastrodia species and their allogamous sisters exhibit genetic profiles typical of self-pollinating plants. This pattern suggests that their ancestors, probably bearing allogamous flowers, had already evolved mechanisms to mitigate the deleterious effects of selfing, potentially facilitating the emergence of complete cleistogamy through benefits such as reproductive assurance, enhanced colonization ability and species reinforcement. Meanwhile, further analyses suggest that complete cleistogamy evolved very recently (possibly within the last 1000-2000 years) in these two Gastrodia lineages. Combined with the scant evidence of complete cleistogamy outside Gastrodia, our findings imply a limited and ephemeral role for complete cleistogamy in plant speciation.

Biological Evolution↗
PubMed2025

Alternative splice acceptor site in MSH4 gene is responsible for male sterility conferred by ms5 in soybean.

In soybean breeding, using the recessive male-sterile ms5 gene, derived from fast neutron mutagenesis, for recurrent selection is advantageous because of the d2 locus, which controls cotyledon color in mature seeds and can be used as a phenotypic selection marker for ms5 male sterility. However, occasional self-fertilization occurs because of the elimination of d2 linkage and instability of male sterility. Elucidating the mechanism and the gene responsible for ms5 male sterility may resolve these problems. Using fine mapping with 15 simple sequence repeat (SSR) markers, we narrowed down the candidate ms5 locus to a 54-kbp region. Bulked-DNA analysis using next-generation sequencing revealed a deletion as a candidate variation in the region. This 15-bp deletion and a nucleotide substitution were identified in intron 1 of MutS homolog (GmMSH4), which modulates chromosomal recombination in meiosis. The ms5 transcript contained a novel exon with a premature termination codon. This exon originated from an alternative splice acceptor site caused by the deletion and nucleotide substitution, disrupting gene function. Co-segregation of male sterility with five independent mutations in GmMSH4 was confirmed using progeny of mutant lines. Mutations in GmMSH4 led to biased DNA partitioning during meiosis, resulting in collapsed or enlarged pollen and suggesting that ms5 male sterility is caused by the failure of pollen formation during meiosis due to the loss of function of GmMSH4. These findings could help explain the mechanism of instability of ms5 male sterility and improve the efficiency of recurrent selection using DNA markers in soybean breeding.

Glycine max↗
PubMed2024

Comparative analysis of chloroplast genomes in ten holly (Ilex) species: insights into phylogenetics and genome evolution.

In order to clarify the chloroplast genomes and structural features of ten Ilex species and provide insights into the phylogeny and genome evolution of the genus Ilex, we conducted a comparative analysis of chloroplast genomes using bioinformatics methods. The chloroplast genomes of ten Ilex species were obtained, and their structural features and variations were compared. The results indicated that all chloroplast genomes in the genus Ilex exhibit a double-stranded circular structure, with sizes ranging from 157,356 to 158,018 bp, showing minimal differences in size. The chloroplast genomes of the ten Ilex species have a relatively conservative gene count, with a total of 134 to 135 genes, including 88 or 89 protein-coding genes, and a conserved number of 8 rRNA genes. Each chloroplast genome contains 3 to 123 SSR (Simple Sequence Repeat) sites, predominantly composed of mononucleotide and trinucleotide repeats, with no detection of pentanucleotide or hexanucleotide repeats. The variation in dispersed repeat sequences among Ilex species is minimal, with a total repeat sequence number ranging from 1 to 14, concentrated in the length range of 30 to 42 base pairs. The expansion and contraction of chloroplast genome boundaries among Ilex species are relatively stable, with only minor variations observed in individual species. Variations in non-coding regions are more pronounced than those in coding regions, with the variability in the Large Single Copy region (LSC) being the highest, while the variability in the Inverted Repeat region A (IRa) is the lowest. The divergence time among Ilex species was estimated using the MCMC-tree module, revealing the evolutionary relationships among these species, their common ancestors, and their differentiation throughout the evolutionary process. The research findings provide a valuable reference for the systematic study and molecular marker development of Ilex plants.

Genome, Chloroplast↗
PubMed2016

Genomic heterozygosity and hybrid breakdown in cotton (Gossypium): different traits, different effects.

BACKGROUND: Hybrid breakdown has been well documented in various species. Relationships between genomic heterozygosity and traits-fitness have been extensively explored especially in the natural populations. But correlations between genomic heterozygosity and vegetative and reproductive traits in cotton interspecific populations have not been studied. In the current study, two reciprocal F2 populations were developed using Gossypium hirsutum cv. Emian 22 and G. barbadense acc. 3-79 as parents to study hybrid breakdown in cotton. A total of 125 simple sequence repeat (SSR) markers were used to genotype the two F2 interspecific populations. RESULTS: To guarantee mutual independence among the genotyped markers, the 125 SSR markers were checked by the linkage disequilibrium analysis. To our knowledge, this is a novel approach to evaluate the individual genomic heterozygosity. After marker checking, 83 common loci were used to assess the extent of genomic heterozygosity. Hybrid breakdown was found extensively in the two interspecific F2 populations particularly on the reproductive traits because of the infertility and the bare seeds. And then, the relationships between the genomic heterozygosity and the vegetative reproductive traits were investigated. The only relationships between hybrid breakdown and heterozygosity were observed in the (Emian22 × 3-79) F2 population for seed index (SI) and boll number per plant (BN). The maternal cytoplasmic environment may have a significant effect on genomic heterozygosity and on correlations between heterozygosity and reproductive traits. CONCLUSIONS: A novel approach was used to evaluate genomic heterozygosity in cotton; and hybrid breakdown was observed in reproductive traits in cotton. These findings may offer new insight into hybrid breakdown in allotetraploid cotton interspecific hybrids, and may be useful for the development of interspecific hybrids for cotton genetic improvement.

Chromosomes, Plant↗
PubMed2026

Strong phylogenetic signal from chloroplast genomes of three Barringtonia species provides the first genomic resources for their conservation.

BACKGROUND: The genus Barringtonia (Lecythidaceae) is a vital component of tropical coastal forests and mangrove ecosystems. Among its members, B. racemosa and B. fusicarpa are classified as Endangered and Vulnerable, respectively, due to habitat degradation and anthropogenic pressures, underscoring the urgent need for genetic studies to guide conservation. Chloroplast (cp.) genomes serve as essential resources for phylogenetic reconstruction and conservation genetics. However, the scarcity of cp. genome data for Barringtonia has limited comprehensive evolutionary and conservation-oriented investigations. RESULTS: We assembled and annotated the first complete cp. genomes of B. racemosa, B. fusicarpa, and B. acutangula. All three genomes exhibit the typical quadripartite structure, ranging from 158,959 bp (B. racemosa) to 159,837 bp (B. acutangula), and contain 132 genes (87 protein-coding, 37 tRNA, 8 rRNA) with a GC content of 36.68%-36.86%. Collinearity and IR boundary analyses revealed high structural conservation without large-scale rearrangements. Interspecific sequence-level variations were detected in simple sequence repeats (SSRs) and long repeats. Nucleotide diversity (π) analysis identified highly polymorphic regions, including rpl20 (π = 0.080), rpoA (π = 0.064), rps3 (π = 0.063), and ndhF (π = 0.060), which represent promising molecular markers for population genetics within the genus. Codon-based selection analyses (Ka/Ks) showed that all protein-coding genes are under strong purifying selection (mean Ka/Ks 0.32-0.37), with no evidence of positive selection. Pairwise genetic distances (p-distances) among Barringtonia species are extremely low (mean 0.0046), while distances to the related genus Bertholletia are ~ 6-fold higher, supporting their generic distinction. CONCLUSIONS: Phylogenetic analysis robustly supports Barringtonia as a monophyletic clade (bootstrap = 100%), with B. racemosa and B. fusicarpa forming a sister lineage to B. acutangula. This study provides the first high-quality cp. genome resources for the two threatened Barringtonia species, revealing strong structural and sequence conservation but no direct chloroplast genomic correlates of endangerment. The identified polymorphic regions and repeat markers lay a foundation for future population genetics, phylogeographic studies, and conservation-oriented genetic management of these ecologically important coastal plants.

Genome, Chloroplast↗
PubMed2024

Nanopore-based full-length transcriptome sequencing for understanding the underlying molecular mechanisms of rapid and slow progression of diabetes nephropathy.

BACKGROUND: Diabetic nephropathy (DN) has been a major factor in the outbreak of end-stage renal disease for decades. As the underlying mechanisms of DN development remains unclear, there is no ideal methods for the diagnosis and therapy. OBJECTIVE: We aimed to explore the key genes and pathways that affect the rate progression of DN. METHODS: Nanopore-based full-length transcriptome sequencing was performed with serum samples from DN patients with slow progression (DNSP, n = 5) and rapid progression (DNRP, n = 6). RESULTS: Here, transcriptome proclaimed 22,682 novel transcripts and obtained 45,808 simple sequence repeats, 1,815 transcription factors, 5,993 complete open reading frames, and 1,050 novel lncRNA from the novel transcripts. Moreover, a total of 341 differentially expressed transcripts (DETs) and 456 differentially expressed genes (DEGs) between the DNSP and DNRP groups were identified. Functional analyses showed that DETs mainly involved in ferroptosis-related pathways such as oxidative phosphorylation, iron ion binding, and mitophagy. Moreover, Functional analyses revealed that DEGs mainly involved in oxidative phosphorylation, lipid metabolism, ferroptosis, autophagy/mitophagy, apoptosis/necroptosis pathway. CONCLUSION: Collectively, our study provided a full-length transcriptome data source for the future DN research, and facilitate a deeper understanding of the molecular mechanisms underlying the differences in fast and slow progression of DN.

Humans↗
PubMed2026

Genome-Wide Identification of SSR and InDel Markers and Experimental Validation of SSR Markers for Distinguishing Cold-Tolerant and Cold-Sensitive Lily Cultivars.

In this study, whole-genome resequencing was performed on the cold-tolerant variety ND-6 and the cold-sensitive variety 'Sorbonne'. After evaluation, the Lilium davidii var. unicolor reference genome was selected to analyze SSR distribution characteristics. Whole-genome InDel identification and comparative analysis were conducted for the two varieties, yielding 34,812,909 and 24,497,857 InDels, respectively. Short InDels were predominant, with deletions slightly outnumbering insertions, mostly located in intergenic regions. Twenty pairs of SSR primers were screened and synthesized. Among them, 10 pairs amplified clearly, with a polymorphism rate of 82.6%, effectively distinguishing the two cultivars examined in this study. This study provides systematic data and a reliable marker resource for the analysis of lily genomic variation, laying a foundation for the identification of cold-tolerant germplasm; validation across additional cultivars and individuals will be required to extend their utility to broader germplasm.

cold resistant lilies↗
PubMed1979

Satellite Ic: a possible link between the satellite DNAs of D. virilis and D. melanogaster.

In this study, we isolated and characterized a previously undetected cryptic satellite DNA comprising 0.1% of the total nuclear genome of D. virilis. This satellite is hidden from detection in neutral CsCl by satellite I and is therefore designated cryptic satellite I or Ic. Sequence analysis reveals that Ic is the repeating heptanucleotide [poly d(AATATAG): d(CTATATT)]. It is more closely related to the three simple sequence satellite DNAs of D. melanogaster, a distantly related species, than it is to any of the major D. virilis satellite DNA sequences. Ic may therefore be a link between the simple sequence satellites of D. virilis and D. melanogaster. As an extension of this theory, we have constructed a "family tree" linking the satellites of D. virilis and D. melanogaster by a series of "simple" operations. Only one intermediate required by this evolutionary scheme has not yet been identified.

Animals↗
PubMed2025

Molecular epidemiological surveillance for non-tuberculous mycobacterial pulmonary disease: a single-center prospective cohort study.

UNLABELLED: Bacterial species cultured from sputum change during treatment or observation for non-tuberculous mycobacterial pulmonary disease; however, strain-level changes remain unrecognized. Variable number tandem repeat typing is a standard technique for strain identification; nonetheless, its labor-intensive and time-consuming nature limits routine clinical use. Therefore, we aimed to elucidate species-subspecies and strain dynamics in non-tuberculous mycobacteria and develop a simple sequence-based strain-level determination method. We performed a single-center prospective cohort study of 112 patients with non-tuberculous mycobacterial pulmonary disease. Whole-genome sequencing was performed on two sputum samples collected at enrollment and at the end of follow-up, followed by variable number tandem repeat (VNTR) typing. We also developed a simple long-read sequencing-based digital VNTR (dVNTR) typing method and evaluated its efficacy. Our results demonstrate that core genome multi-locus sequencing typing revealed species/subspecies changes in 13 patients (11.6%); VNTR typing detected strain changes in 16 patients (14.3%) without species/subspecies changes. Overall, pathogen shifts occurred in 29 patients (shift [+] group, 25.9%), whereas 83 had no detectable pathogen shift (shift [-] group, 74.1%). Interestingly, macrolide and amikacin susceptibility changed in both groups, but resistance remained higher in shift (-) patients. dVNTR results aligned with those of conventional VNTR typing. In conclusion, since susceptibility factors remain unclear, routine species/subspecies identification and molecular typing, such as VNTR, are optimal for patient care. Core genome multi-locus sequencing typing with a dVNTR identified pathogen shifts, innovating non-tuberculous mycobacterial pulmonary disease management.Clinical TrialsThis study is registered with UMIN as UMIN 000056067. IMPORTANCE: Pulmonary non-tuberculous mycobacterial disease is a chronic infection in which the causative pathogens may change at the species, subspecies, or strain level over time. Accurate tracking of these changes is essential for optimizing treatment; however, conventional clinical practice lacks efficient methods for monitoring such dynamics. Our study revealed pathogen changes in approximately one-quarter of patients over 1.5 years, prompting the development of a novel surveillance system that integrates next-generation sequencing for both species-subspecies identification and strain-level molecular epidemiology. This innovation enables real-time monitoring of pathogen dynamics, allowing clinicians to promptly adjust treatment strategies and improve patient care through more informed decision-making.

Humans↗
PubMed2026

mm2-ivh: simple and precise overlap detection in alpha satellite HORs with interval hashing.

SUMMARY: We propose a new algorithm, "interval hashing," which distinguishes identical k-mers arising from different repeat sequences, particularly in complex repeat arrays such as alpha satellite HORs. We implement this algorithm as a fork of minimap2, named mm2-ivh. In local assembly of alpha satellite HORs, mm2-ivh accurately reconstructs more haplotypes than assemblers using standard minimizers. AVAILABILITY AND IMPLEMENTATION: mm2-ivh is available under the MIT license at https://github.com/ocxtal/mm2-ivh and runs on common Unix-compatible systems.

Algorithms↗
PubMed1979

Subunit structure of alpha-satellite DNA containing chromatin from African green monkey cells.

alpha-Satellite DNA containing chromatin from African green monkey cells (CV-1 cells) has been used to study the question whether or not nucleosomes are arranged in phase with the 172 bp repeat unit of the satellite DNA. Digestion experiments with DNAase II led us to exclude a simple phase relationship between the nucleosomal and the satellite DNA repeats. Digestion of CV-1 nuclei with micrococcal nuclease and endogenous nuclease (s) produced a series of sharp bands in the satellite DNA register over a background of heterogeneous length fragments. This observation is explained by a preferential cleavage of certain nucleotide sequences by these nucleases and is not in contradiction to our conclusion that a simple phase relationship does not exist.

Animals↗
PubMed2026

Spacer-engineered donor DNA enhances CRISPR-Cas9-mediated knockin to establish a chemical knockdown platform for endogenous proteins.

Precise installation of functional protein domains at endogenous loci is a powerful approach for interrogating protein functions, but its broad application is limited by the low efficiency of homology-directed repair (HDR)-mediated knockin during Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing. Here, we investigated a simple donor DNA engineering strategy that enhances HDR-mediated gene knockin by appending additional gRNA-recognizable spacer sequences to donor templates. Systematic analysis of linear dsDNA and plasmid donors showed that spacer position, length, and orientation influenced HDR efficiency, and that spacer-containing donors improved knockin across multiple genomic loci, insertion sizes, cell types and delivery modalities. Mechanistic analyses revealed that spacer-containing donors formed stable complexes with Cas9/gRNA and showed increased nuclear localization, supporting nuclear delivery as a key contributor to improved editing outcomes. We then applied this gene-editing strategy to establish a chemical knockdown platform by installing drug-responsive degrons at endogenous loci, generating cell lines in which GSK3β or Lin28A protein could be rapidly, potently and reversibly depleted by drug treatment. These platforms enable selective modulation of endogenous proteins and reveal cellular responses that may differ from those obtained using conventional genetic perturbation. Together, this work establishes a readily implementable framework that integrates improved gene editing with on-demand chemical knockdown of endogenous proteins.

CRISPR-Cas9↗
PubMed2025

Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.

UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings. IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.

Isoniazid↗
PubMed2026

High-Content CRISPR Screening: Methods and Applications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening has become a central technology in functional genomics, enabling genome-scale interrogation via pooled perturbations. Early CRISPR screens employed survival or simple phenotypic readouts to identify essential genes and drug resistance mechanisms. However, as biological questions have shifted toward understanding regulatory networks, cellular heterogeneity, and context-dependent gene functions, there has been increasing demand for screening strategies capable of capturing complex cellular phenotypes beyond cell fitness. Recent advances in single-cell sequencing, high-content imaging, and spatial transcriptomics have expanded the resolution of CRISPR screening by enabling multidimensional phenotypic characterization following genetic perturbation. By integrating pooled perturbations with diverse readouts, these approaches systematically map targeted gene edits to transcriptional states, cellular phenotypes, and microenvironmental contexts. Meanwhile, innovations in library design, delivery, and computational pipelines have further improved the robustness and interpretability of high-content screening platforms. This review synthesizes the methodological evolution of CRISPR screening, emphasizing advances in perturbation strategies, delivery systems, and multimodal readouts. Representative applications spanning oncology, immunotherapy, developmental biology, neurobiology, and infectious diseases are delineated to demonstrate refined gene network annotations. Additionally, existing technical bottlenecks, such as scalability, cost constraints, and in vivo limitations, are critically assessed. Finally, future directions are proposed to facilitate the development of precise medicine.

CRISPR screening↗