利用核酸和真核溶酶MOC1进行可编程,精确的双链DNA断裂生成
Gundra Sivakrishna Rao1, Ahmed H Saleh1, Firdaws Melliti1
1Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia.
Analytical chemistry
|February 1, 2024
概括
我们开发了一种新的基因编辑技术,称为PNA辅助Resolvase介导 (PNR) 编辑. 这种方法使用核酸 (PNA) 和一种特定的酶来精确地在目标位置切割DNA.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 遗传学 是一个遗传学.
背景情况:
- 可编程特定位点核酶 (SSN) 对于基因编辑至关重要,但面临着传递和特异性挑战.
- 目前的基因组工程技术通常依赖于结构引导的核酶,但成功程度有限.
- 对于更广泛的应用,需要更小,更具体的SSN.
研究的目的:
- 开发一种使用核酸 (PNA) 和真核细胞溶酶的新型基因编辑技术.
- 为了证明PNA辅助的Resolvase介导 (PNR) 编辑在体外的效率和特异性.
- 优化PNR编辑条件并探索其多重分离能力.
主要方法:
- 利用核酸 (PNA) 进行特定地点的DNA入侵.
- 采用了来自Arabidopsis thaliana的真核分解酶AtMOC1进行DNA裂变.
- 进行了体外实验,以测试PNR编辑概念,特异性和切割部位映射.
- 优化反应条件,包括温度,缓冲和裂变时间.
- 研究了PNA类型,长度和周围核酸序列的作用.
主要成果:
- 证明了PNR编辑在体外的精确目标特异性.
- 对于AtMOC1-介导的裂变,围绕PNA入侵确定了核酸要求.
- 绘制了AtMOC1.1生成的特定裂变点的地图.
- 展示了用于精确释放DNA片段的多重裂变.
- 建立了PNA入侵和AtMOC1裂变的最佳体外条件.
结论:
- PNR编辑为特定地点的DNA裂变提供了一个有希望的新方法.
- 该技术具有很高的特异性和多重应用的潜力.
- 优化 PNA 设计和反应条件是有效的 in vitro 分裂的关键.
- 编辑PNR解决了当前基因组工程技术的一些局限性.
相关概念视频
02:31Homologous Recombination
50.5K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
50.5K
02:04Fixing Double-strand Breaks
12.6K
The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
12.6K
01:20Mismatch Repair
4.8K
Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
4.8K
02:37Restarting Stalled Replication Forks
5.8K
DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
5.8K
02:53Conservative Site-specific Recombination and Phase Variation
6.0K
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...
6.0K
01:08Nucleotide Excision Repair
37.1K
Overview
37.1K