The exploration of pathogenic single nucleotide polymorphisms in the genome plays a pivotal role in the study of human disease-associated genetic mutations. However, there remains a lack of suitable high-throughput screening platforms to investigate the impact of point mutations on genomic structure and function. CRISPR/Cas9-mediated base editors has enabled large-scale annotation of the human genome and phenotypic characterization of monogenic disorders. Base editors, a precise gene-editing technique capable of achieving targeted base substitutions, can be employed to induce mutations at specific functional sites, thereby observing their effects on gene expression, protein function, and cellular phenotypes. Furthermore, integrating base editors with high-throughput screening technologies allows for large-scale evaluation of multiple candidate sites, accelerating the identification of functional loci and providing a powerful tool for disease research and therapeutic target discovery. This article aims to introduce the working principles of various base editors, including cytosine base editors, adenine base editors, and prime editors, and summarize recent advances in high-throughput screening of functional genomic sites using base-editing techniques.
Humans ; Gene Editing/methods* ; CRISPR-Cas Systems/genetics* ; Genome, Human ; Polymorphism, Single Nucleotide

This article reviews the review articles and research papers related to biomanufacturing driven by engineered organisms published in the Chinese Journal of Biotechnology from 2023 to 2024. The content covers 26 aspects, including chassis cells; gene (genome) editing; facilities, tools and methods; biosensors; protein design and engineering; peptides and proteins; screening, expression, characterization and modification of enzymes; biocatalysis; bioactive substances; plant natural products; microbial natural products; development of microbial resources and biopesticides; steroidal compounds; amino acids and their derivatives; vitamins and their derivatives; nucleosides; sugars, sugar alcohols, oligosaccharides, polysaccharides and glycolipids; organic acids and monomers of bio-based materials; biodegradation of polymeric materials and biodegradable materials; intestinal microorganisms, live bacterial drugs and synthetic microbiomes; microbial stress resistance engineering; biodegradation and conversion utilization of lignocellulose; C1 biotechnology; bioelectron transfer and biooxidation-reduction; biotechnological environmental protection; risks and regulation of biomanufacturing driven by engineered organisms, with hundreds of technologies and products commented. It is expected to provide a reference for readers to understand the latest progress in research, development and commercialization related to biomanufacturing driven by engineered organisms.
Biotechnology/methods* ; Gene Editing ; Genetic Engineering ; Metabolic Engineering ; Protein Engineering ; Biosensing Techniques

The OsSPL10 gene has previously been reported to positively regulate trichome development and negatively regulate salt and drought stress tolerance in rice. However, it is not clear whether this gene can be used for gene editing to create new germplasm of glabrous leaf and salt-tolerant rice. In this study, we created six rice mutants by CRISPR/Cas9-mediated editing of OsSPL10 from 'Xinfeng 2', 'Xinkedao 31', and 'Xindao 25', the main rice cultivars along the Yellow River. Visual observation and scanning electron microscopy verified that the mutants lacked trichomes on the leaves and glumes, and the expression of glabrous marker genes OsHL6, OsGL6, and OsWOX3B in mutants was down-regulated compared with that in the wild type. The net photosynthetic rate, stomatal conductance, and transpiration rate of flag leaves in the mutants were significantly higher than those in the wild type. In addition, the survival rates of the mutants were much higher than that of the wild type after 7 days of treatment with 200 mmol/L NaCl. The results of quantitative real-time polymerase chain reaction (qRT-PCR) further verified that compared with the wild type, the mutants demonstrated down-regulated expression of the salt stress-related gene OsGASR1 and up-regulated expression of OsNHX2 and OsIDS1. Statistical analysis of agronomic traits showed that the mutants had increased plant height and no significant changes in yield-related traits compared with the wild type. The six spl10 mutants created in this study not only had glabrous leaves and glumes but also demonstrated enhanced tolerance to salt stress, serving as new germplasm resources for directional breeding of rice along the Yellow River.
Oryza/physiology* ; CRISPR-Cas Systems/genetics* ; Salt Tolerance/genetics* ; Gene Editing/methods* ; Plant Proteins/genetics* ; Rivers ; Plant Leaves/genetics* ; Mutation ; Plants, Genetically Modified/genetics* ; China

With the rapid advancement of synthetic biology, CRISPR-Cas systems have emerged as a powerful tool for gene editing, demonstrating significant potential in various fields, including medicine, agriculture, and industrial biotechnology. This review comprehensively summarizes the significant progress in applying artificial intelligence (AI) technologies to the design, mining, and modification of CRISPR-Cas systems. AI technologies, especially machine learning, have revolutionized sgRNA design by analyzing high-throughput sequencing data, thereby improving the editing efficiency and predicting off-target effects with high accuracy. Furthermore, this paper explores the role of AI in sgRNA design and evaluation, highlighting its contributions to the annotation and mining of CRISPR arrays and Cas proteins, as well as its potential for modifying key proteins involved in gene editing. These advancements have not only improved the efficiency and precision of gene editing but also expanded the horizons of genome engineering, paving the way for intelligent and precise genome editing.
CRISPR-Cas Systems/genetics* ; Artificial Intelligence ; Gene Editing/methods* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Machine Learning ; Humans ; Genetic Engineering/methods* ; Synthetic Biology

Nucleic acid elements are essential functional sequences that play critical roles in regulating gene expression, optimizing pathways, and enabling gene editing to enhance the production of target products in biomanufacturing. Therefore, the design and optimization of these elements are crucial in constructing efficient cell factories. Artificial intelligence (AI) provides robust support for biomanufacturing by accurately predicting functional nucleic acid elements, designing and optimizing sequences with quantified functions, and elucidating the operating mechanisms of these elements. In recent years, AI has significantly accelerated the progress in biomanufacturing by reducing experimental workloads through the design and optimization of promoters, ribosome-binding sites, terminators, and their combinations. Despite these advancements, the application of AI in biomanufacturing remains limited due to the complexity of biological systems and the lack of highly quantified training data. This review summarizes the various nucleic acid elements utilized in biomanufacturing, the tools developed for predicting and designing these elements based on AI algorithms, and the case studies showcasing the applications of AI in biomanufacturing. By integrating AI with synthetic biology and high-throughput techniques, we anticipate the development of more efficient tools for designing nucleic acid elements and accelerating the application of AI in biomanufacturing.
Artificial Intelligence ; Synthetic Biology ; Nucleic Acids/genetics* ; Algorithms ; Gene Editing ; Promoter Regions, Genetic ; Biotechnology/methods*

DNA double-strand breaks represent a common type of serious DNA damage in living organisms, causing instability of the genome and leading to cell death. Homologous recombination and non-homologous end-joining (NHEJ) are the two main ways to repair DNA double-strand breaks. The core components involved in the NHEJ pathway are highly conserved in both yeast and humans. A few bacteria such as Mycobacterium, Pseudomonas aeruginosa, and Bacillus subtilis also have the NHEJ mechanism. NHEJ plays a key role in the double strand repair of Mycobacterium in latency. This paper summarizes the mechanism and important components of NHEJ in Mycobacterium, introduces the application of NHEJ in gene editing, and reviews the research progress of the NHEJ pathway in Mycobacterium. We hope to bring new insights into the molecular mechanism and provide clues for the application of NHEJ in Mycobacterium.
DNA End-Joining Repair/physiology* ; Gene Editing/methods* ; Mycobacterium/physiology* ; DNA Breaks, Double-Stranded ; Humans

Conventional cancer therapies, such as radiotherapy and chemotherapy, often damage normal cells and may induce new tumors. Oncolytic viruses (OVs) selectively target tumor cells while sparing normal cells. Most OVs used in clinical trials have been genetically engineered to enhance their ability to target tumor cells and activate immune responses. To develop a specific OV-based approach for treating cervical cancer, this study constructed an oncolytic adenovirus that delivered a base editor targeting oncogenes to achieve efficient killing of tumor cells through inhibiting tumor growth and directly lysing tumor cells. We utilized the human telomerase reverse transcriptase (TERT) promoter to drive the expression of adenovirus early region 1A (E1A) and successfully constructed the P-hTERT-E1A-GFP vector, which was validated for its activity in cervical cancer cells. Given the critical role of the MYC oncogene in the research of oncology, identifying efficient editing sites for the MYC oncogene is a key step in this study.Three MYC-targeting gRNAs were engineered and co-delivered with ABE8e base editor plasmids into HEK293T cells. Following puromycin selection, Sanger sequencing demonstrated differential editing efficiencies: MYC-1 (43%), MYC-2 (25%), and MYC-3 (35%), identifying MYC-1 as the most efficient editing locus. By constructing the P-ABEs-hTERT-E1A-GFP and P-MYC gRNA-hTERT-E1A-GFP vectors, we successfully packaged the virus and confirmed its specificity and efficacy. The experimental results demonstrate that this novel oncolytic adenovirus effectively inhibits the growth of HeLa cells in vitro, providing new experimental evidence and potential strategies for treating cervical cancer based on the HeLa cell model.
Humans ; Uterine Cervical Neoplasms/pathology* ; Oncolytic Viruses/genetics* ; Female ; HEK293 Cells ; Oncolytic Virotherapy/methods* ; Adenoviridae/genetics* ; Gene Editing/methods* ; Telomerase/genetics* ; Adenovirus E1A Proteins/genetics* ; Genetic Vectors/genetics* ; HeLa Cells

Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.
CRISPR-Cas Systems/genetics* ; Gene Editing/methods* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

: Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.
CRISPR-Cas Systems/genetics* ; Gene Editing/methods* ; Bacteroides/genetics* ; Humans ; Gastrointestinal Microbiome/genetics* ; Bacteroides fragilis/genetics*

The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant in vitro model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating Sox17 in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate Sox17 in sESCs. The expression of Sox17 was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest Sox17 expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.
Animals ; CRISPR-Cas Systems/genetics* ; Sheep ; SOXF Transcription Factors/genetics* ; Embryonic Stem Cells/cytology* ; Genetic Vectors/genetics* ; Cell Differentiation/genetics* ; Transfection ; Gene Editing/methods*