强大的微型基于Cas的转录调制通过工程Un1Cas12f1和连接Sso7d
Xiangnan Wang1, Lingyun Li1, Li Guo2
1Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Molecular therapy : the journal of the American Society of Gene Therapy
|February 14, 2024
概括
研究人员开发了miniCRa和miniCRi,小型的CRISPR-Cas12f系统用于基因激活和沉默. 这些系统包装在腺相关病毒 (AAV) 载体中,显示出用于基因治疗应用的高效率.
科学领域:
- 分子生物学分子生物学
- 基因编辑技术的技术
- 生物技术是生物技术.
背景情况:
- 克里斯普尔-Cas12f系统提供了一个小型基因编辑工具,适用于腺相关病毒 (AAV) 传递.
- 现有的Cas12f系统在转录调制中表现出可变的效率.
- 基因治疗需要强大而高效的微型CRISPR系统.
研究的目的:
- 设计一个高效的基于CRISPR的微型转录激活系统 (miniCRa).
- 开发一个强大的微型基于CRISPR的转录沉默系统 (miniCRi).
- 通过使用AAV向量来评估这些系统的治疗潜力.
主要方法:
- 对Un1Cas12f1核酶和引导RNA进行工程,以创建激活的miniCRa.
- 将Sso7dDNA结合蛋白与miniCRa融合以增强活性 (SminiCRa).
- 将突变引入Un1Cas12f1以产生miniCRi和SminiCRi用于压制.
- 开发一个全集的AAV载体 (AIOminiCRi),用于体内基因沉默.
主要成果:
- miniCRa实现了ASCL1基因激活的1319倍增加.
- SminiCRa显示ASCL1基因的激活为5,628倍,以及其他基因的显著激活.
- miniCRi和SminiCRi在8个测试基因中平均实现了80%的抑制.
- 在Huh-7细胞中,AIOminiCRi AAVs有效抑制了70%的SERPINA1基因.
结论:
- miniCRa和SminiCRa是高效基因转录激活的强大工具.
- miniCRi和SminiCRi提供强大和特定的基因沉默能力.
- 这些微型CRISPR系统适合用于AAV包装,并有望用于生物医学研究和基因治疗.
相关概念视频
RNA Polymerase II Accessory Proteins
9.2K
Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
9.2K
Cooperative Binding of Transcription Regulators
6.4K
Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome. Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form...
6.4K
Combinatorial Gene Control
8.3K
Combinatorial gene control is the synergistic action of several transcriptional factors to regulate the expression of a single gene. The absence of one or more of these factors may lead to a significant difference in the level of gene expression or repression.
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
8.3K
Co-activators and Co-repressors
7.4K
Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
7.4K
Master Transcription Regulators
6.9K
Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
6.9K


