Nature Protocols
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2014
·Vol. 9
·期 10
·2493-2512
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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
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实验动物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2021
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IF 6.558
Yanju Chen,Hui Wu,Siwenjie Qian,Xiaoping Yu,Huan Chen,Jian Wu
Heavy metal lead accumulation in the environment pollutes the ecology systems and further threatens the human health. It is necessary to develop a sensitive method to detect it. Here, we propose a highly sensitive lead detection method by combining DNAzyme and CRISPR system. Once the lead ion is recognized, the substrate chain of DNAzyme is cleaved to produce single strand DNA . The produced single strand DNA can be detected by Cas protein/guide RNA complex and further trigger the collateral cleavage effect of CRISPR system, which can indiscriminately cut short single strand DNA reporters. By this way, the detection signals can be greatly amplified. This method can detect lead ions as low as 0.48 nM. The sensitivity is higher than the DNAzyme method. Furthermore, the portable 3D printing device is designed to observe the fluorescent signals so the end-point detection results can be visualized by the naked eyes. The entire detection process can avoid using bulky and expensive instruments, which can promote on-site lead ion detection.
研究领域:
生物化学
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
INFECTION GENETICS AND EVOLUTION
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2015
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IF 3.015
Qinqin Huang,Hongping Luo,Minqiang Liu,Jie Zeng,Abualgasim Elgaili Abdalla,Xiangke Duan,Qiming Li,Jianping Xie
Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea . CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements . Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis . The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2.
研究领域:
微生物学
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免疫学
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分子生物学
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DOI
高影响因子
Advanced Science
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2025
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IF 14.1
Yating Li,Jason Tan,Yanqin Tu,Jingnan Wu,Zaifang Zhang,Yifeng Wei,Xinan Jiao,Yan Zhou
DNA base modification is a common strategy used by bacteriophages to evade host immune detection. A prominent example is dU-DNA, where thymidine is globally replaced with 2′-deoxyuridine. Despite its widespread occurrence, dU-DNA's biosynthetic pathways and functional roles remain incompletely understood. Here, different enzymes supporting dU-DNA biosynthesis in phage PBS1, Roseophage DSS3_VP1, and Yersiniophage PhiR1-37 are identified and characterized. The dU nucleotide precursor is supplied by phage dCTP deaminases (Dcds). Thymidine nucleotides are degraded by dTMP phosphatases (Dtms) in PBS1 and DSS3_VP1, and by a dTTP pyrophosphatase (Dtt) in PhiR1-37, preventing incorporation into phage DNA. The dU-DNA, isolated from Yersiniophage PhiR1-37 or synthesized by PCR, demonstrates resistance to cleavage by restriction enzymes recognizing thymidine-containing sequences, partial resistance to Lb Cas12a nuclease recognizing a TTTV PAM site, while remaining sensitive to Sp Cas9 nuclease recognizing a NGG PAM site. A phylogenetic analysis of PBS1 dCTP deaminase and closely related T4 phage dCMP deaminase suggest possible evolutionary origins from bacterial dCDP deaminases. Overall, these findings suggest independent acquisition of dU-DNA biosynthetic enzymes and pathways in the diverse phages, and support its protective function against different host-encoded nucleases.
研究领域:
分子生物学
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病毒学
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微生物学
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基因工程
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核酸代谢
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免疫逃避
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进化生物学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2025
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IF 6
Jingzhi Zhou,Xichen Shi,Chaoqin He,Xiuwen Zheng,Ruo Yuan,Xia Yang
Background Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO 2 , its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO 2 is suboptimal. Results In this study, lychee-like Fe–TiO 2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO 2 , facilitating exciton resonance. The amount of Fe in TiO 2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88 fmol/L. Significance This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.
研究领域:
生物传感器
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纳米技术
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生物化学
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材料科学
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分子生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
Microbiology Spectrum
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2024
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IF 3.7
Yun Li,Ruifeng Ma,Chenying Gao,Ziyi Li,Yongqin Zheng,Fang Fang,Cheng Wang,Guohua Li,Xiaozhen Du,Changbao Xu,Meirong Xu,Rui Liu,Xiaoling Deng,Zheng Zheng
“Candidatus Liberibacter asiaticus” (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars (“Shatangju” mandarin, “Guanxi” pomelo, and “Shatian” pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth.
研究领域:
植物病理学
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分子生物学
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代谢组学
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转录组学
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DOI
PubMed
高影响因子
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