在哺乳动物细胞中进行dCas13介导的转化抑制,以实现精确的基因沉默
Antonios Apostolopoulos1,2, Naohiro Kawamoto2, Siu Yu A Chow3
1Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan.
Nature communications
|March 12, 2024
概括
研究人员开发了CRISPRδ,这是一种新型的基因沉默方法,可以在不降解mRNA的情况下阻止翻译. 这种高度特定的方法为RNA干扰和CRISPR-Cas13系统提供了一个精确的基因淘汰替代方案.
科学领域:
- 分子生物学分子生物学
- 基因法规 基因法规
- 生物技术是生物技术.
背景情况:
- 目前的基因沉默方法,如RNA干扰 (RNAi) 和CRISPR-Cas13,具有局限性,包括非目标效应和附带mRNA裂变.
- 需要更具体,更有效的基因淘汰策略.
研究的目的:
- 开发一种具有增强特异性和减少非目标效应的新型基因沉默工具.
- 建立一种使用催化无活性Cas13 (dCas13) 的转化沉默方法.
主要方法:
- 采用CRISPRδ系统,利用触媒不活性的Cas13 (dCas13) 蛋白和指导RNA准起始编码子.
- 在不同翻译启动机制 (依赖上限,IRES,RAN) 中评估翻译沉默的有效性.
- 基因组范围内的核糖体分析,以评估基因沉默特异性.
主要成果:
- 克里斯普尔δ通过作为核糖体的物理障碍来有效地沉默基因转换.
- 当指导RNA准起始编码子时,无论翻译启动机制如何,都观察到高沉默效率.
- 全基因组的核糖体分析证实了CRISPRδ的超高特异性,对mRNA稳定性的影响最小.
- dCas13与转化压缩器的融合进一步提高了沉声性能.
结论:
- 克里斯普尔提供了一种高特异性和有效的方法,通过抑制翻译来破坏基因.
- 这种方法克服了现有的RNAi和CRISPR-Cas13系统的局限性.
- 在真核生物系统中,CRISPRδ提供了一个基于转化抑制的基因沉默的多功能框架.
相关概念视频
Experimental RNAi
6.1K
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
6.1K
Regulation of Expression at Multiple Steps
906
The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the...
906
MicroRNAs
3.0K
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
3.0K
siRNA - Small Interfering RNAs
16.8K
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
16.8K
Regulation of Expression Occurs at Multiple Steps
22.7K
Gene expression can be regulated at almost every step from gene to protein. Transcription is the step that is most commonly regulated. This involves the binding of proteins to short regulatory sequences on the DNA. This association can either promote or inhibit the transcription of a gene associated with the respective sequence.
Transcription results in the generation of precursor (pre-mRNA) that consists of both exons and introns, which needs further processing before being translated to a...
Transcription results in the generation of precursor (pre-mRNA) that consists of both exons and introns, which needs further processing before being translated to a...
22.7K
Nonsense-mediated mRNA Decay
10.6K
The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
10.6K


