MOLECULAR AND CELLULAR BIOCHEMISTRY
·
2023
·
IF 4.3
Lu Junnan,Tong Ying,Dong Rui,Yang Yijun,Hu Wen,Zhang Minghong,Liu Quan,Zhao Siting,Adams John H.,Qin Li,Chen Xiaoping
CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce “marker-free” genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.
研究领域:
寄生虫学
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分子生物学
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基因组编辑
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DOI
PubMed
高影响因子
MOLECULAR CELL
·
2020
·
IF 15.584
Shuai Jin,Hongyuan Fei,Zixu Zhu,Yingfeng Luo,Jinxing Liu,Shenghan Gao,Feng Zhang,Yu-Hang Chen,Yanpeng Wang,Caixia Gao
Summary Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.
研究领域:
分子生物学
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遗传学
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DOI
PubMed
高影响因子
Genes
·
2025
·Vol. 16
·期 2
·217
·
IF 2.8
Haiyan Shu,Aiping Luan,Hidayat Ullah,Junhu He,You Wang,Chengjie Chen,Qing Wei,Rulin Zhan,Shenghe Chang
Background/Objectives: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications. Methods: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted. Results: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5′-NGG (“N” represents any nucleobase, followed by two guanine “G”) PAM sites at the 3′ end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5′-NGG PAM site at the 3′ end. Conversely, although the target sequence with a 5′-NAG PAM site (where “N” is any nucleobase, followed by adenine “A” and guanine “G”) adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5′-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1,AcOT5,AcCSPE6,AcPKG11A), more deletions than insertions were found. Conclusions: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.
CAS / MDL:
研究领域:
植物学
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遗传学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Biotechnology Journal
·
2024
·Vol. 19
·期 11
·e202400527
·
IF 3.2
Chaoqun Tang,Lu Zhang,Junyi Wang,Congjia Zou,Yalin Zhang,Jifeng Yuan
Saccharomyces boulardii , as a probiotic yeast, has shown great potential in regulating gut health and treating gastrointestinal diseases. Due to its unique antimicrobial and immune-regulating functions, it has become a significant subject of research in the field of probiotics. In this study, we aim to enhance the antioxidant properties of S. boulardii by producing l -ergothioneine (EGT). We first constructed a double knockout of ura3 and trp1 gene in S. boulardii to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii , we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii . By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. By using PiggyBac transposon system, a great variety of EGT-producing strains was obtained, which is not possible for the conventional single target genome editing, and one best isolated top producer reached 17.50 mg/L EGT after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.
研究领域:
微生物学
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遗传工程
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营养学
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产品关联
DOI
PubMed
高影响因子
Nature Protocols
·
2014
·Vol. 9
·期 10
·2493-2512
·
IF 7.782
Shao Yanjiao,Guan Yuting,Wang Liren,Qiu Zhongwei,Liu Meizhen,Chen Yuting,Wu Lijuan,Li Yongmei,Ma Xueyun,Liu Mingyao,Li Dali
Conventional embryonic stem cell (ESC)–based gene targeting, zinc-finger nuclease (ZFN) and transcription activator–like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
研究领域:
分子生物学
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遗传学
·
实验动物学
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产品关联
DOI
PubMed
高影响因子
BIOMEDICINE & PHARMACOTHERAPY
·
2022
·
IF 7.419
Qingxia Zuo,Wanqing Xu,Yanbin Wan,Dongyan Feng,Changsheng He,Cailing Lin,Dongchao Huang,Feng Chen,Liya Han,Qi Sun,Dong Chen,Hongli Du,Lizhen Huang
CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.
研究领域:
药理学
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分子生物学
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基因工程
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产品关联
DOI
PubMed
高影响因子
Biology-Basel
·
2023
·Vol. 12
·期 10
·1331
·
IF 4.2
Xincheng Zhang,Fang Wang,Mi Ou,Haiyang Liu,Qing Luo,Shuzhan Fei,Jian Zhao,Kunci Chen,Qingshun Zhao,Kaibin Li
Simple SummaryMyostatin is a negative regulator of muscle growth and development, and mutational inactivation of this gene increases muscle mass. Knockout of themstnbgene inPelteobagrus fulvidracodid not increase muscle mass, although our previous study of themstnagene did. Instead, it reduced the body size and growth performance ofPelteobagrus fulvidraco. Through the present study, we found that the mutantPelteobagrus fulvidracohad a reduction in the number of vertebrae, the length, and the intervertebral distance inPelteobagrus fulvidraco, and these changes may be the underlying cause of the shorter body length in mutantPelteobagrus fulvidraco. Histological comparison of the same sites in themstnmutant and wild groups ofPelteobagrus fulvidracoalso revealed that the number and density of osteocytes were greater inmstnbknockoutPelteobagrus fulvidracothan in wild-typePelteobagrus fulvidraco.AbstractBased on obtainingmstnbgene knockout inPelteobagrus fulvidraco, a study on the effect of themstngene on skeletal morphology and growth was performed by comparing the number and length of the vertebrae of mutant and wild-type fish in a sibling group ofP. fulvidraco, combined with the differences in cells at the level of vertebral skeletal tissue. It was found thatmstnbgene knockdown resulted in a reduction in the number of vertebrae, the length, and the intervertebral distance inP. fulvidraco, and these changes may be the underlying cause of the shorter body length in mutantP. fulvidraco. Further, histological comparison of the same sites in themstnmutant and wild groups ofP. fulvidracoalso revealed that the number and density of osteocytes were greater inmstnbknockoutP. fulvidracothan in wild-typeP. fulvidraco. Our results demonstrated that when using genome editing technology to breed new lines, the effects of knockout need to be analyzed comprehensively and may have some unexpected effects due to insufficient study of the function of certain genes.Keywords:myostatin b;genome editing;Pelteobagrus fulvidraco;body length;skeleton;vertebral centrum
研究领域:
水产养殖学
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分子生物学
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遗传学
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2023
·
IF 8.8
Hao Luo,Yihong Bao,Ping Zhu
Nutritional and functional soy-based milk gains growing attention globally in food industry. However, its poor sensorial attributes, single flavor, and limited substance variety become critical issues in displaying balanced nutrition and multifunction for health. Herein, a novel probiotic lycopene-soy milk was developed by genome edited Bacillus subtilis harboring lycopene biosynthesis cassette with efficient lycopene production of 25.73 ± 1.57 mg/g DCW. Further investigation displayed desirable pH, reducing sugar, protein, total phenolic content and isoflavone for achieved milk than conventional soy milk , implying it with well-balanced nutritional quality . Notably, achieved milk exhibited stronger antioxidant capacity and higher isoflavone bioavailability for functionality. Moreover, it possessed significantly high scores for taste, appearance, and overall acceptability, suggesting its excellent sensorial attributes. To our delight, it is the first time to fortify soy-milk with probiotic and lycopene by genome edited B. subtilis to explore additive effect on improving nutritional value and functionality for food application.
研究领域:
食品科学
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微生物学
·
营养学
✓ 已验证
DOI
PubMed
高影响因子
Nature Communications
·
2025
·Vol. 16
·期 1
·1-15
·
IF 15.7
Liu Zhen,Wu Jiacai,Wang Ning,Lin Yongqi,Song Ruiteng,Zhang Min,Li Bin
Despite remarkable progress in designing RNA delivery systems, endosomal escape remains a recognized challenge for efficient RNA delivery. In this study, we develop a robust mRNA delivery platform termed endosomolytic chloroquine-like optimized lipid nanoparticles (ecoLNPs) for versatile mRNA delivery in vitro and in vivo via integrating the signature scaffold extracted from endosomolytic chloroquine into ionizable lipids. RNase-resistant ecoLNPs are capable of delivering a broad variety of mRNA payloads to diverse cell types, even hard-to-transfect 3D cells, with an efficiency of up to 18.9-fold higher than that of commercial transfection reagents. The pH-responsive endosomolytic activity of ecoLNPs can be largely attributed to the proton sponge effect and saposin B-promoted membrane disruption. In vivo, ecoLNPs enable potent local and systemic mRNA delivery and exhibit comparable potency to the clinically approved mRNA vaccine carrier, but strong tropism for lymph nodes following intramuscular injection. Furthermore, ecoLNPs are able to retain in vivo delivery potency for at least one week under non-frozen conditions and induce efficient genome editing in transgenic mice. Overall, the structure-guided integration strategy provides a pathway for de novo design of endosomolytic mRNA delivery systems.
研究领域:
分子生物学
·
纳米医学
·
药物递送
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产品关联
DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
·
2024
·
IF 4.4
Bing Fu,Meng Chen,Xianfeng Bao,Jiajie Lu,Zhiwen Zhu,Fuyao Guan,Chuyang Yan,Peize Wang,Linglin Fu,Ping Yu
Vitamin B 2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.
研究领域:
生物工程
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
Bioengineered
·
2022
·
IF 6.832
Wei-Hao Wu,Xiao-Mei Ma,Jian-Qing Huang,Qin Lai,Fu-Neng Jiang,Cui-Yun Zou,Long-Tian Chen,Lian Yu
This study investigated whether CRISPR/Cas9 (D10A) nickase-mediated gene editing can correct the aberrant Hb Constant Spring mutation (Hb CS or HBA2: c.427 T > C) in fibroblasts. Vectors for repairing the α-globin-encoding gene, HBA2:c.427 T > C mutation, includingthe CRISPR/Cas9(D10A)-sg plasmid and donor with homology arms, were constructed and used to perform gene editing in patient-derived fibroblasts. We subsequently analyzed the genetic correction, the gene editing efficiency and off-target effect. Sequencing analysis and the BamHI assay showed that HB CS mutant cells were repaired with Hb CS point mutations, the editing efficiency was 4.18%~9.34% and no off-target effects were detected. The results indicate that the HB CS mutant gene is effectively repaired by the CRISPR/Cas9 (D10A)system, which may enable truly personalized therapy for precise repair of α-thalassemia.
研究领域:
分子生物学
·
遗传学
·
医学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF PHARMACEUTICS
·
2024
·
IF 5.3
Qian Sun,Hongqian Zhang,Feng Ding,Xue Gao,Zongwei Zhu,Chuanxu Yang
CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.
研究领域:
分子生物学
·
基因治疗
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纳米医学
✓ 已验证
DOI
PubMed
高影响因子
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