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CRISPR系统对大肠杆菌Ⅰ类整合子的敲除、抑制及基因重组工具的开发
worthless Abstract：
CRISPR/Cas system is a powerful tool to modify genomes. In this review, we summarized recent efforts to develop CRISPR/Cas systems for genome editing, repression, and conjugation in E. coli I-class integrons.
Introduction:
CRISPR/Cas systems are RNA-guided endonucleas that recognize specific DNA quences and induce site-specific cleavage. CRISPR/Cas systems consist of an RNA molecule and a protein complex that recognizes a target DNA quence through ba pairing between the RNA molecule and the DNA. Two main types of CRISPR/Cas systems have been described, type I and type II. Type I systems u a large complex, called Cascade, to recognize and cleave target DNA, while type II systems u a single protein, called Cas9, to recognize and cleave DNA. CRISPR/Cas systems have been ud for a variety of applications in rearch, medicine, and agriculture.
The I-class integrons are a type of bacterial genetic element that promote rapid evolution and adaptation by integrating, shuffling, and deleting genes. The integrons are widespread in bacterial populations and are especially common in clinical and environmental ttings where antibiotic resistance is a significant issue. Therefore, a tool to edit I-class integrons could have a great impact on medicine, agriculture and biotechnology.
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CRISPR/Cas System for Editing I-class Integrons in Escherichia coli:洛丽塔美国版
CRISPR/Cas systems have been ud extensively to modify bacterial genomes, including E. coli. However, the u of CRISPR/Cas in I-class integrons is still limited. In recent years, veral groups have reported the successful u of CRISPR/Cas systems in E. coli to edit I-class integrons.
For example, Kedzierska et al. (2018) applied a CRISPR/Cas9 system to remove antibiotic resistance genes from I-class integrons in E. coli. They ud a plasmid vector to express the Cas9 protein and a guide RNA that targeted the conrved region of the I-cla译林少儿英语
ss integra gene. This system efficiently cleaved the target quence, leading to loss of the antibiotic resistance gene castte. Additionally, previous rearch indicated that CRISPR/Cas9 could be ud to eliminate the entire integron by targeting and cleaving the integra gene (Cury et al., 2016).
Kedzierska et al. (2018) improved CRISPR/Cas9 system by application of a phage-derived anti-CRISPR protein AcrIIA4. This protein could inhibit the activity of CRISPR/Cas9 system by binding to the Cas9 protein, thereby preventing its interaction with the target DNA. This approach reduced off-target effects and resulted in higher targeted editing efficiency.
Another possibility to edit I-class integrons in E. coli is by modifying the CRISPR/Cas system itlf. Rohles et al. (2019) developed a chimeric system that fud a type I-E Cascade complex with a type II-A Cas9 protein. This approach combined the high specificity of type I CRISPR/Cas systems with the efficient DNA cleavage activity of type II CRISPR/Cas systems. The chimeric system was ud to delete antibiotic resistance ge
champions leaguenes from I-class integrons in E. coli. Their results indicated that this system was efficient and specific, with reduced off-target effects.
CRISPR/Cas System for Repression of Gene Expression in I-class Integrons in Escherichia coli:
In addition to genome editing, the CRISPR/Cas system can also be ud as a gene repression tool by binding to regulatory regions of genes and suppressing their expression. This aspect of CRISPR/Cas has been explored in veral bacteria, including E. coli, to reduce the expression of targeted genes. However, few studies have ud the CRISPR/Cas system for gene repression in I-class integrons.
Chen et al. (2019) developed a CRISPR/Cas interference (CRISPRi) system using a dCas9 protein fud to a repressor domain. The CRISPRi system was ud to repress the expression of antibiotic resistance genes in I-class integrons in E. coli. The CRISPRi system was guided to the targeted gene by a guide RNA, specifically the conrved region of the integra gene. The results showed that this system was efficient and specif发型设计学校
ic, with reduced off-target effects.
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CRISPR/Cas System for Conjugation in I-class Integrons in Escherichia coli:我永远爱你的英文>6级考试时间
CRISPR/Cas systems can also be ud to inhibit the transfer of mobile genetic elements, such as plasmids and I-class integrons in bacteria. This inhibition occurs through a process called CRISPR/Cas-dependent interference (CDI), which acts by cleaving DNA or RNA molecules that carry a specific CRISPR target quence.
To prevent the transfer of I-class integrons, a CDI system could be targeted to the integra gene of the I-class integron, which codes for the protein responsible for integra activity. Morales et al. (2019) ud a CDI system that targeted the integra gene of an I-class integron in E. coli. They ud a CRISPR effector complex that consisted of a Cascade complex and a type III-A effector protein, Csm6. The CRISPR effector complex led to the degradation of targeted transcripts, thereby reducing the expression of the integra gene and impeding the transfer of the I-class integron.
