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Beyond Cutting: CRISPR-Driven Synthetic Biology Toolkit for Next-Generation Microalgal Metabolic Engineering.
Microalgae, with their unparalleled capabilities for sunlight-driven growth, CO2 fixation, and synthesis of diverse high-value compounds, represent sustainable cell factories for a circular bioeconomy. However, industrial deployment has been hindered by biological constraints and the inadequacy of conventional genetic tools. The advent of CRISPR-Cas systems initially provided precise gene editing via targeted DNA cleavage. This review argues that the true transformative potential lies in moving decisively beyond cutting to harness CRISPR as a versatile synthetic biology "Swiss Army Knife". We synthesize the rapid evolution of CRISPR-derived tools-including transcriptional modulators (CRISPRa/i), epigenome editors, base/prime editors, multiplexed systems, and biosensor-integrated logic gates-and their revolutionary applications in microalgal engineering. These tools enable tunable gene expression, stable epigenetic reprogramming, DSB-free nucleotide-level precision editing, coordinated rewiring of complex metabolic networks, and dynamic, autonomous control in response to environmental cues. We critically evaluate their deployment to enhance photosynthesis, boost lipid/biofuel production, engineer high-value compound pathways (carotenoids, PUFAs, proteins), improve stress resilience, and optimize carbon utilization. Persistent challenges-species-specific tool optimization, delivery efficiency, genetic stability, scalability, and biosafety-are analyzed, alongside emerging solutions and future directions integrating AI, automation, and multi-omics. The strategic integration of this CRISPR toolkit unlocks the potential to engineer robust, high-productivity microalgal cell factories, finally realizing their promise as sustainable platforms for next-generation biomanufacturing.
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Development of a prime editor with improved editing efficiency in Arabidopsis.
Prime editing is widely used in many organisms to introduce site-specific sequence modifications such as base substitutions, insertions, and deletions in genomic DNA without generating double-strand breaks. Despite wide-ranging applications of prime editing, prime editors (PEs) have low editing efficiencies, especially in dicot plants. Therefore, PEs are barely used for genome engineering in dicot plant species. Here, based on previous approaches used to improve prime editing efficiency, we generated different combinations of PE components and prime editing guide RNAs (pegRNAs) and examined their prime editing efficiencies in Arabidopsis thaliana protoplasts as a dicot model system. We found that v4e2, in which PE was fused to viral nucleocapsid (NC) protein, RNase Hdeleted M-MLV RT, and a dominant negative version of human mutL homolog 1 (hMLH1dn), showed the highest prime editing efficiency in Arabidopsis protoplasts when it was co-transfected with dual enhanced pegRNA. Our results suggest that the v4e2 PE system could be used for efficient prime editing in dicot plants. [BMB Reports 2025; 58(2): 70-74].
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Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs.
Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.
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Gene editing unlocks superior mutants from once detrimental RFL for enhanced rice yield traits.
RICE FLORICULA LEAFY/ABERRANT PANICLE ORGANIZATION 2 (RFL/APO2) is a master regulator of panicle morphogenesis and development in rice. Traditionally, mutations in RFL have led to severe growth phenotypes and decreased rice yield, labeling it as detrimental. However, the present study challenged this perception by utilizing CRISPR/Cpf1 and single-base gene-editing technologies to generate a series of site-directed rfl mutants. Our findings revealed that the evolutionarily conserved sterile alpha motif (SAM) domain and DNA-binding domain (DBD), as well as the intron region of RFL, all play roles in regulating rice morphological development and yield traits. Specifically, introns and the SAM domain are primarily involved in panicle development, whereas the DBD and its key functional sites are closely associated with morphological development and yield. Notably, the amino acid at position 266 was found to be a critical site for RFL regulation of grain shape, significantly affecting grain weight, with changes in the expression levels of genes involved in grain length and panicle weight regulation, such as GRF1 and SPL16. This study not only expands our understanding of the role of RFL in monocot plants but also provides a novel perspective on how gene editing can transform a gene once considered detrimental to improve yield traits in cereal crops. These findings suggest that the number of genes available for optimizing rice phenotypes through gene editing can be significantly increased.
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Somatic Gene Editing of GUCY2D by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque.
Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.
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An adenine base editor variant expands context compatibility.
Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the target A is in a TA context. Here we evolve the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA (R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a Streptococcus pyogenes Cas9 nickase, expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in PCSK9, whose disruption reduces low-density lipoprotein cholesterol, and ABCA4-p.Gly1961Glu, the most frequent mutation in Stargardt disease.
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New cytosine base editor for plant genome editing.
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Application and progress of CRISPR/Cas9 gene editing in B-cell lymphoma: a narrative review.
BACKGROUND AND OBJECTIVE: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) gene editing and CRISPR/Cas9 screening libraries are hot topics, and have high application values in the diagnosis and treatment of genetic diseases, and the improvement of prognosis. The major treatment of B-cell lymphoma is chemotherapy combined with biological therapy. Due to the individual specificity and the emergence of drug resistance, the therapeutic efficacy varies. The objective of this article is to explore potential targets to enhance therapeutic effects, optimize treatment plans, and improve the prognosis of patients with B-cell lymphoma. METHODS: We undertook a comprehensive, narrative review of the latest literature to define the current application and progress of CRISPR/Cas9 in B-cell lymphoma. KEY CONTENT AND FINDINGS: The concepts of CRISPR/Cas9, the mechanism of gene editing, and the procedures of CRISPR/Cas9 screening libraries are investigated for candidate genes. We mainly focus on application and progress of CRISPR/Cas9 in B-cell lymphoma and screen out some genes, signaling pathways, and cytokines, which may become potential targets for clinical treatment. CONCLUSIONS: CRISPR/Cas9 gene editing has great promise in the treatment of B-cell lymphoma. This article reviews some genes, signaling pathways, and cytokines related to the progression and prognosis of B-cell lymphoma to provide a strong theoretical basis.
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Prime Editing: The Next Frontier in Precision Gene Therapy.
Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.
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Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants.
CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA. In both protocols, we used single-stranded oligonucleotides as a repair template for homologous recombination and insertion of stop codons in the target gene.
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