SEARCH · PubMed Health

Results for “CRISPR-cas9 screening”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed2025

An in vivo barcoded CRISPR-Cas9 screen identifies Ncoa4-mediated ferritinophagy as a dependence in Tet2-deficient hematopoiesis.

TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4-dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.

CRISPR-Cas Systems↗

PubMed2026

CRISPR-Cas9 screen to identify genes regulating cell death.

Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Understanding the mechanisms of cell death is crucial for elucidating the control of cellular homeostasis and developing therapies for related diseases. Despite extensive research efforts spanning decades, many aspects of cell death mechanisms remain elusive, highlighting the need for continued exploration. Here, we describe how to identify novel regulators involved in cell death pathways using a genome-wide screening approach.

CRISPR-Cas Systems↗

PubMed2026

In vivo CRISPR screening links NFKB1 to endocrine resistance in ER+ breast cancer.

Resistance to endocrine therapy (ET) remains a major clinical challenge in the treatment of estrogen receptor-positive (ER+) breast cancer, underscoring the need for novel therapeutic targets. To identify genetic drivers of ET resistance, we conducted an in vivo genome-wide CRISPR-Cas9 screen in MCF7 cells implanted into ovariectomized nude mice under estrogen-deprived conditions. Despite the bottlenecks inherent to in vivo pooled screening, recurrent enrichment analysis identified NFKB1 as a candidate regulator of estrogen-independent tumor progression. Functional studies confirmed that NFKB1 deficiency enhanced tumorigenicity and conferred resistance to tamoxifen and fulvestrant both in vitro and in vivo. Mechanistically, transcriptomic and biochemical analyses revealed that NFKB1 deficiency activated canonical NF-κB signaling, leading to inflammatory gene induction and enhanced ER signaling. Furthermore, pharmacologic inhibition of NF-κB signaling restored ET sensitivity in NFKB1-deficient cells. Analysis of TCGA breast cancer datasets revealed reduced NFKB1 expression in luminal breast tumors, whereas expression of other NF-κB family members was largely unchanged. Further analysis showed that low NFKB1 expression was associated with poorer clinical outcomes in patients with ER+ breast cancer. Collectively, these findings identify NFKB1 as a negative regulator of NF-κB signaling and endocrine resistance in ER+ breast cancer and provide mechanistic evidence linking NF-κB activation to ligand-independent ER signaling. Our results support further investigation of NFKB1 as a candidate biomarker and of NF-κB pathway inhibition as a potential therapeutic strategy in endocrine therapy-resistant breast cancer. These findings also illustrate the utility of in vivo CRISPR screening for identifying candidate regulators of endocrine resistance in breast cancer.

ER+ breast cancer↗

PubMed2026

MYDGF as a telomerase activator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) involves progressive cartilage breakdown. This is driven by impaired chondrocyte function and an imbalance in the extracellular matrix homeostasis. However, the key upstream regulators that maintain healthy chondrocytes are still not fully known. Here, we report that myeloid-derived growth factor (MYDGF) is a novel regulator of telomerase activity and is critical for cartilage health. Using a genome-wide CRISPR-Cas9 screen with a TERT reporter system, MYDGF was identified as a strong positive regulator of telomerase. Functional studies confirmed that MYDGF positively regulates TERT expression and telomerase activity in both HeLa and ATDC5 cells. In a mouse surgical destabilization of the medial meniscus (DMM) OA model, MYDGF knockout (KO) mice exhibited more severe cartilage damage. Conversely, delivering MYDGF into the knee joint using AAV virus partially alleviated cartilage injury. Transcriptome profiling revealed that MYDGF-deficient chondrocytes exhibit downregulated key pathways involved in matrix building and upregulated inflammatory signals, indicating a shift in cell state. In summary, our work establishes MYDGF as a key upstream factor for both telomerase and cartilage matrix homeostasis remodeling. It connects telomerase function to chondrocyte health. These findings also highlight MYDGF as a promising new target for treating osteoarthritis.

Cartilage degeneration↗

PubMed2025

CDK12 inhibition reveals melanoma dependence on the RUNX1/CBFβ complex for genomic stability.

Cutaneous melanoma is the deadliest form of skin cancer, frequently driven by hyperactivation of the RAS/mitogen-activated protein kinase (MAPK) pathway. Cyclin-dependent kinase 12 (CDK12), a downstream effector of MAPK signaling, has emerged as a therapeutic target due to its essential role in transcriptional regulation and DNA damage repair. To identify vulnerabilities associated with CDK12 inhibition, we performed a genome-wide CRISPR-Cas9 screen and identified the Runt-related transcription factor RUNX1 and its cofactor CBFβ as synthetic lethal partners of CDK12. RUNX1 inhibition enhanced melanoma sensitivity to CDK12 inhibitors in a p53-independent manner, resulting in DNA damage accumulation and impaired repair capacity. Combined inhibition of CDK12 and RUNX1 suppressed melanoma growth in vivo. These findings identify RUNX1/CBFβ as a compensatory mechanism in CDK12-inhibited melanoma and define a synthetic lethal interaction with translational potential for combinatorial therapy.

Core Binding Factor Alpha 2 Subunit↗

PubMed2026

FANCM is required for the PAX3::FOXO1-driven oncogenic program in rhabdomyosarcoma.

Many cancers are driven by mutationally altered transcription factors (TFs) that rewire cells to an oncogenic state. Cells must activate specific mechanisms to tolerate the burden of oncogenic TF activity. To define such mechanisms, we focused on a canonical oncogenic fusion protein-driven cancer, alveolar rhabdomyosarcoma (ARMS), where the PAX3::FOXO1 fusion protein hyperactivates and mislocalizes PAX3 and FOXO1 TF functions. Employing sequential functional genomic CRISPR-Cas9 screens, we identified FANCM, a DNA translocase in the Fanconi anemia pathway, as a selective dependency in PAX3::FOXO1+ ARMS. FANCM loss reduces fusion protein levels, induces myogenic differentiation, and disrupts the PAX3::FOXO1 transcriptional program, thereby halting oncogenic proliferation. Mechanistically, FANCM depletion exacerbates replication stress (RS) and DNA damage signaling, with chromatin-associated RS enriched at PAX3::FOXO1 target gene loci, resulting in selective downregulation of the oncogenic program. CRISPR exon-tiling screens prioritized FANCM's helicase and DNA-binding domains as essential for this dependency, linking FANCM-mediated replication fork binding to sustained oncogenesis.

ARMS↗

PubMed2025

Inferring metabolic objectives and trade-offs in single cells during embryogenesis.

While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning. We validated SCOOTI by identifying essential genes from CRISPR-Cas9 screens in embryonic stem cells, and by inferring the metabolic objectives of quiescent cells, during different cell-cycle phases. Applying this to embryonic cell states, we observed a decrease in metabolic entropy upon development. We further uncovered a trade-off between glutathione and biosynthetic precursors in one-cell zygote, two-cell embryo, and blastocyst cells, potentially representing a trade-off between pluripotency and proliferation. A record of this paper's transparent peer review process is included in the supplemental information.

Single-Cell Analysis↗

PubMed2026

An SLC7A5-dependent nutrient-sensing circuit overcomes cisplatin tolerance via mTOR-autophagy signaling.

Cisplatin-based chemotherapy responses are highly heterogeneous across cancers, with the mechanisms governing drug sensitivity remaining incompletely understood. Using genome-wide CRISPR-Cas9 knockout screening, we systematically characterized regulators of cisplatin response and uncovered a counterintuitive finding: mTOR inhibition promotes cisplatin tolerance, contradicting the canonical view that PI3K-AKT-mTOR activation confers chemoresistance. Mechanistically, both mTOR suppression and cisplatin treatment converge to activate cytoprotective autophagy, which enhances cancer cell survival under therapeutic stress. The amino acid transporter SLC7A5 was identified and validated as a key integrator of the mTOR-autophagy axis that modulates cisplatin sensitivity. SLC7A5 expression positively correlates with cisplatin sensitivity across cancer cell lines, and its downregulation is associated with cisplatin resistance in multiple cancer types, supporting its potential as a mechanistically grounded predictive biomarker. Translationally, leucine supplementation sensitizes cancer cells to cisplatin in an SLC7A5-mTOR-autophagy-dependent manner. Collectively, our study defines a novel mTOR-autophagy adaptive loop governing cisplatin tolerance, positions SLC7A5 as a central regulatory node with both biomarker and therapeutic target value, and proposes leucine supplementation as a simple, translatable strategy to improve cisplatin efficacy in SLC7A5-expressing tumors.

Cisplatin↗

PubMed2024

A novel molecular pathway of lipid accumulation in human hepatocytes caused by PFOA and PFOS.

Exposed to ubiquitously perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) has been associated with non-alcoholic fatty liver disease (NAFLD), yet the underlying molecular mechanism remains elusive. The extrapolation of empirical studies correlating per- and polyfluoroalkyl substance (PFAS) exposure with NAFLD occurrence to real-life exposure was hindered by the limited availability of mechanistic data at environmentally relevant concentrations. Herein, a novel pathway mediating hepatocyte lipid accumulation by PFOA and PFOS at human-relevant dose (<10 μM) was identified by integrating CRISPR-Cas9 genome screening, concentration-dependent transcriptional assay in HepG2 cell and epidemiological data mining. 1) At genetic level, nudt7 showed the highest enriched potency among 569 NAFLD-related genes, and the transcription of nudt7 was significantly downregulated by PFOA and PFOS exposure (<7 μM). 2) At molecular pathway, upon exposure to ≤10-4 μM PFOA and PFOS, the downregulation of nudt7 transcriptional expression triggered the reduction of Ace-CoA hydrolase activity. 3) At cellular level, increased lipids were measured in HepG2 cells with PFOA and PFOS (<2 μM). Overall, we identified a novel mechanism mediated by transcriptional downregulation of nudt7 gene in hepatocellular lipid increase treated with PFOA and PFOS, which could potentially explain the NAFLD occurrence associated with exposure to PFASs in humans.

Humans↗

PubMed2026

Genetic and biochemical screens identify MGAT1 as a druggable glycosyltransferase target in STK11-mutant lung cancer.

Checkpoint inhibitors are standard-of-care therapies for non-small cell lung cancer (NSCLC), but their efficacy is limited in tumors with STK11 mutations, highlighting the need for new therapeutic strategies. Here, we performed complementary in vivo and in vitro CRISPR-Cas9 functional genomic screens to identify genes whose loss restores sensitivity to anti-PD-1 therapy. We found that loss of MGAT1, a Golgi glycosyltransferase critical for the maturation of high-mannose N-glycans into hybrid and complex glycan structures, reversed resistance to anti-PD-1 treatment in syngeneic mouse tumor models harboring STK11 mutations. Parallel co-culture screens with antigen-matched CD8+ T cells further showed that disruption of N-glycosylation strongly sensitized tumor cells to T cell-mediated killing. Genetic rescue studies demonstrated that this immune-evasion phenotype depends on MGAT1 catalytic activity, supporting direct biochemical interrogation of the enzyme. Using purified human MGAT1 and a UDP-Glo™ glycosyltransferase assay, we established a tractable screening platform and performed a 500,000-compound biochemical high-throughput screen, identifying an initial hit (compound 1; IC50 = 197 μM). Subsequent medicinal chemistry optimization delivered progressively more potent analogs, including TNG-9333 (0.814 μM) and TNG-2673 (0.043 μM) and represented a >1000-fold improvement in biochemical potency from the starting hit. Crystal structures of human MGAT1 in apo, UDP-bound, UDP-GlcNAc-bound, and inhibitor-bound states, together with SPR and DSF analyses, revealed that this chemical series engages a previously unrecognized allosteric pocket and inhibits MGAT1 through a UDP-noncompetitive mechanism. Collectively, our work implicates N-glycosylation as a key mediator of immune evasion and establishes MGAT1 as a ligandable, structurally tractable target for small-molecule drug discovery.

CRISPR/Cas9 target discovery↗

PubMed2026

A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.

Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.

Yarrowia↗

PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗

PubMed2026

CRISPGen: A deep generative framework for multi-objective CRISPR/Cas9 guide RNA design via Conditional Latent Diffusion and Dual-Critic Reinforcement Learning.

MOTIVATION: The CRISPR-Cas9 system offers transformative potential for precision genome editing, yet its clinical translation remains constrained by the risk of unintended off-target double-strand breaks. While current discriminative models excel at evaluating pre-specified candidate guides, resolving the fundamental antagonism between on-target cleavage efficiency and off-target specificity within a fixed sequence search space remains a major challenge. RESULTS: We present CRISPGen, a unified deep generative framework that reframes sgRNA design as a multi-objective constrained sequence synthesis problem. It integrates (i) DNABERT-2 genomic-language embeddings, (ii) a conditional latent diffusion generator conditioned on a user-specified on-target efficiency target, and (iii) a dual-critic reinforcement-learning (RL) stage that couples a frozen on-target efficiency critic with a cross-attention off-target discriminator (validation Pearson R=0.8157) trained on a unified corpus of experimental off-target events from six detection platforms. Across 1000 generated sgRNAs, CRISPGen reduces the mean off-target discriminator score by 99.7% relative to the pre-RL baseline and, under an exhaustive whole-genome screen of all 302,631,056 NGG PAM sites in GRCh38, yields zero perfect-match and only 55 one-mismatch genomic hits. We further show, transparently, that the internal on-target critic saturates under RL optimization - an instance of Goodhart's Law - and therefore assess on-target viability using an independent external CRISPRon screen (mean 47.10/100). Repeating the RL fine-tuning stage under three random seeds (with the diffusion generator, DNABERT-2 embeddings, and off-target discriminator held fixed) yields a stable operating point across seeds. Full diversity, per-mismatch, and reproducibility statistics are reported in the Results. AVAILABILITY: Source code is available at https://github.com/malekpouri/CRISPGen; the pre-trained checkpoints and the 3,000,000-sequence library are hosted on Hugging Face (https://huggingface.co/malekpouri/CRISPGen-Checkpoints) and archived on Zenodo under DOI 10.5281/zenodo.21428641.

CRISPR-Cas9↗

PubMed2026

A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.

CRISPR/Cpf1↗

PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗

PubMed2026

Cross-target and cell-preferential CRISPR-Cas9 inhibition with carbohydrate-tagged oligonucleotides.

Precise control of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) activity is important for limiting off-target effects and chromosomal rearrangements. Existing inhibitors, including anti-CRISPR proteins and spacer-targeting oligonucleotides, can be constrained by immunogenicity, target-sequence dependence, or delivery challenges. Here, we developed single-stranded DNA oligonucleotides that target conserved regions of the Streptococcus pyogenes Cas9 single-guide RNA (sgRNA) scaffold rather than the variable spacer. Screening identified single-stranded DNA 7 (ssDNA7), which targets stem-loop-1 and the adjacent linker and inhibits Cas9 activity across multiple tested spacer sequences and genomic loci without redesigning the inhibitor. Carbohydrate conjugation improved oligonucleotide stability and preferentially enhanced inhibitory activity in selected liver-derived or cancer cell models. Amplicon sequencing confirmed inhibition of endogenous editing in multiple cell models and normal liver-derived organoids, and supported cell-preferential inhibition in matched cell comparisons. These findings establish sgRNA-scaffold targeting as a strategy for cross-target inhibition of Cas9 and show that carbohydrate conjugation can tune its cellular activity.

CRISPR-Cas9↗

PubMed2026

Enhanced exonuclease-Cas9 systems promote multiple nucleotide deletions with higher efficiency and broader targeting scope in plants.

CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.

CRISPR-Cas Systems↗

PubMed2026

An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species.

Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. KEY POINTS: • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.

CRISPR-Cas Systems↗