Frontiers in Bioengineering and Biotechnology
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2020
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IF 3.644
Sun Jun,Lu Li-Bing,Liang Tian-Xin,Yang Li-Rong,Wu Jian-Ping
Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25–100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90–100% and 25–35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.
研究领域:
合成生物学
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代谢工程
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微生物遗传学
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生物技术
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
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2022
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IF 0
Meixia Wang,Fang Yan,Huanbin Zhou
Summary Base editing is a precision genome-editing approach that is widely utilized to generate single-nucleotide variants (SNVs) in genomes. Here, we present a protocol to perform targeted adenine (A)-to-guanine (G) substitution in rice using adenine base editor (ABE). We detail the design of sgRNA, CRISPR plasmid construction, rapid genetic transformation of rice, and genotyping of editing events. This protocol can be applied to cytosine base editing in rice as well. For complete details on the use and execution of this protocol, please refer to Yan et al. (2021). 1
研究领域:
植物生物学
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基因组编辑
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分子遗传学
✓ 已验证
DOI
PubMed
BIOMATERIALS
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2023
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IF 14
Quan Zou,Yi Lu,Bo Qing,Na Li,Ting Zhou,Jinbin Pan,Xuejun Zhang,Xuening Zhang,Yupeng Chen,Shao-Kai Sun
CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.
研究领域:
遗传学
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分子生物学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Microbial Cell Factories
·
2022
·Vol. 21
·期 1
·1-13
·
IF 6.352
Xin Qinglong,Chen Yudan,Chen Qianlin,Wang Bin,Pan Li
Background Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus . This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02. Results In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes ( aprE , nprE , wprA , and bamHIR ) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain. Conclusions The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.
研究领域:
微生物学
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基因工程
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生物技术
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DOI
PubMed
高影响因子
Nature Protocols
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2020
·Vol. 16
·期 1
·431-457
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IF 10.419
Jin Shuai,Gao Qiang,Gao Caixia
Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12–15 weeks.
研究领域:
遗传学
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植物生物学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Cell Host & Microbe
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2022
·
IF 31.316
Zhen-Ping Zou,Yue Du,Ting-Ting Fang,Ying Zhou,Bang-Ce Ye
Summary Rapid advances in synthetic biology have fueled interest in engineered microorganisms that can diagnose and treat disease. However, designing bacteria that detect dynamic disease-associated biomarkers that then drive treatment remains difficult. Here, we have developed an engineered probiotic that noninvasively monitors and records inflammatory bowel disease (IBD) occurrence and progression in real time and can release treatments via a self-tunable mechanism in response to these biomarkers. These intelligent responsive bacteria for diagnosis and therapy (i-ROBOT) consists of E. coli Nissle 1917 that responds to levels of the inflammatory marker thiosulfate by activating a base-editing system to generate a heritable genomic DNA sequence as well as producing a colorimetric signal. Fluctuations in thiosulfate also drive the tunable release of the immunomodulator AvCystatin . Orally administering i-ROBOT to mice with colitis generated molecular recording signals in processed fecal and colon samples and effectively ameliorated disease. i-ROBOT provides a promising paradigm for gastrointestinal and other metabolic disorders.
研究领域:
合成生物学
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微生物学
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胃肠病学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
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2023
·
IF 0
Zhen-Ping Zou,Yu-Hang Fan,Yue Du,Ting-Ting Fang,Wei Du,Ying Zhou,Bang-Ce Ye
Summary Engineered microorganisms hold potential for disease diagnosis and treatment. Here, we present a protocol to engineer E. coli Nissle 1917 strain (iROBOT) using genome insertion and plasmid construction to diagnose, record, and ameliorate inflammatory bowel disease in mice. We describe steps for constructing and administering iROBOT, diagnosing and recording colitis, preparing samples, and analyzing fluorescence and base editing ratios of iROBOT. We detail a colitis ameliorating assay using the disease activity index, colon length, tissue pathological section, and cytokine analysis. For complete details of the use and execution of this protocol, please refer to Zou et al. 1
研究领域:
微生物学
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分子生物学
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基因工程
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炎症性肠病
✓ 已验证
DOI
PubMed
NUCLEIC ACIDS RESEARCH
·
2024
·
IF 16.6
Wu Yaokang,Li Yang,Liu Yanfeng,Xiu Xiang,Liu Jiaheng,Zhang Linpei,Li Jianghua,Du Guocheng,Lv Xueqin,Chen Jian,Ledesma-Amaro Rodrigo,Liu Long
Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.
研究领域:
分子生物学
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基因工程
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微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nature Biomedical Engineering
·
2025
·
IF 26.6
Xu Saijuan,Liang Dan,Wang Qiudao,Cheng Yan,Xie Da,Gui Yang,Zhang Haokun,Feng Changrui,Zhao Feiyan,Ren Wendan,Sun Gongrui,Yang Yang,Li Lin,Lai Yongrong,Fu Bin,Lu Yuming,Wang Zi Jun,Wu Yuxuan
Ex vivo autologous haematopoietic stem cell (HSC) gene therapy provides a promising treatment option for haematological disorders. However, current methods involve complex processes and chemotherapeutic conditioning, leading to limited accessibility for treatment and major side effects. Here we develop antibody-free targeted lipid nanoparticles (LNPs) for mRNA delivery to HSCs in vivo, enabling efficient base editing of the γ-globin gene ( HBG1/2 ) promoter target in human HSCs to reactivate fetal haemoglobin in derived erythroid cells. Delivery of ABE8e/sgRNA mRNA with optimized LNPs achieves efficient in vivo base editing of HBG1/2 in transfusion-dependent β-thalassaemia (TDT) patient-derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. Our research indicates that using LNPs for genome editor delivery achieves efficient editing of endogenous genes of human HSCs. This non-viral delivery system eliminates the need for collecting or mobilizing HSCs, providing a potent and one-time treatment potential for blood disorders such as sickle cell disease and TDT.
研究领域:
遗传学
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分子生物学
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生物技术
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基因治疗
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纳米医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）