对RNA导向转体子的活性和特异性的全面分析揭示了设计改进变体的机会
Seong Guk Park1, Jung-Un Park1,2,3, Esteban Dodero-Rojas1
1Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, United States.
Nucleic acids research
|September 23, 2025
概括
工程化CRISPR相关的转子子 (CAST) 显示了增强的基因组编辑能力. 研究人员开发了一种查方法,以提高CAST活动和治疗应用的特异性.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 与CRISPR相关的转位子 (CAST) 是自然的RNA引导的DNA转位系统.
- 野生类型的CAST在非本土系统中具有有限的集成活动,因此需要用于治疗的工程.
- 开发高效和特定的CAST变体对于推进基因组编辑技术至关重要.
研究的目的:
- 开发一种高通量查方法,以量化CAST变种活动和特异性.
- 通过分析核心蛋白质突变,获得对CAST转换的机制性见解.
- 为了确定增强活动和特异性的CAST突变,改进基因组编辑工具.
主要方法:
- 开发了一种高通量双基因屏幕来评估CAST变体的性能.
- 在核心转位蛋白 (TnsB,TnsC,TniQ) 上使用位点和突变发生.
- 工程 CAST 系统的量化现场定位特异性和集成活动.
主要成果:
- 野生型V-K CAST系统在选条件下显示了88-95%的现场准特异性.
- 突变发生过程揭示了对转位子蛋白功能的机制性见解,并确定了活动特异性的权衡.
- 确定了增加CAST活动高达四倍的特定突变,对向特异性的影响最小.
结论:
- 开发的选平台有效地表征了CAST序列功能关系.
- 已经建立了优化CAST活动和特异性的工程原理.
- 确定了增强的CAST变种,其基因组编辑的治疗潜力得到了改善.
更多相关视频
11:12Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach
Published on: September 11, 2017
7.9K
08:19Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Published on: July 7, 2020
11.3K
相关概念视频
DNA-only Transposons
17.2K
DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...
The donor site from where the transposon is excised is either degraded or...
17.2K
Overview of Transposition and Recombination
18.8K
Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
18.8K
Conservative Site-specific Recombination and Phase Variation
6.7K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
6.7K
Transposons
1.4K
Transposons, or "jumping genes," are small mobile genetic elements (MGEs) that range from 700 to 40,000 base pairs in length. They are found in all organisms and can move within the same chromosome or transfer to different chromosomes. In some cases, transposons can also jump between different host DNA molecules, such as plasmids or viruses, contributing to genetic variability.Barbara McClintock first discovered these mobile genetic elements in the 1940s while studying maize genetics, and she...
1.4K
piRNA - Piwi-interacting RNAs
7.5K
PIWI-interacting RNAs, or piRNAs, are the most abundant short non-coding RNAs. More than 20,000 genes have been found in humans that code for piRNAs while only 2000 genes have been found for miRNAs. piRNAs can act at the transcriptional and post-transcriptional levels and have a vital role in silencing transposable elements present in germ cells. They are also involved in epigenetic silencing and activation. Previously, they were thought to function only in germ cells but new evidence suggests...
7.5K
Ribosome Profiling
4.1K
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
4.1K
