在CRISPR/Cas9诱导的双链断裂中发现精确修复的动态
Daniela Ben-Tov1, Fabrizio Mafessoni1, Amit Cucuy1
1Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel.
Nature communications
|June 14, 2024
概括
由于精确的DNA修复,CRISPR/Cas9基因编辑效率受到限制. 这项研究量化了CRISPR/Cas9切割和修复动态,揭示了高保真性修复显著影响突变发生的结果.
科学领域:
- 分子生物学分子生物学
- 遗传学 遗传学 是一个
- 生物技术是生物技术.
背景情况:
- 通过CRISPR/Cas9技术,通过有针对性的DNA双链断裂 (DSB) 实现精确的基因编辑.
- 了解DSB诱导,易出错的修复和精确修复的速率对于优化CRISPR/Cas9应用至关重要.
- 目前的方法很难全面测量这些修复动态.
研究的目的:
- 开发和验证用于量化CRISPR/Cas9诱导的DSB中间体和修复产品的分子和计算工具包.
- 描述DSB诱导,处理和修复在内源位置的时间动态.
- 阐明精确与容易出错的修复对整体突变发生的贡献.
主要方法:
- 开发基于单分子测序的工具包,用于多重定量化.
- 在番茄原生质中进行DSB诱导和修复动态的时间过程分析 (72小时).
- 动力建模以整合实验数据并推断修复途径的贡献.
主要成果:
- 该工具包成功量化了多个目标的DSB诱导和修复动态.
- 分裂率在64-88%之间,分裂频率在15-41%之间.
- 精确维修占所有维修事件的70%,显著影响了indel积累.
结论:
- 开发的系统有效地解了DSB感应和修复动态.
- 高保真性修复在限制CRISPR介导突变发生效率方面发挥着至关重要的作用.
- 这个工具包提供了一种新的方法来剖析基因组编辑中的DNA修复途径.
相关概念视频
Fixing Double-strand Breaks
12.5K
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...
12.5K
Homologous Recombination
50.4K
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...
50.4K
Long-patch Base Excision Repair
7.0K
Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
7.0K
Restarting 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
Translesion DNA Polymerases
9.9K
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
9.9K
Overview of DNA Repair
31.0K
In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
Chemically...
Chemically...
31.0K


