相关实验视频

Updated: Jun 26, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

一个高保真性DNA酶辅助的CRISPR/Cas13a系统,具有单核酸解决特异性

Yunping Wu¹, Ruigang Jin¹, Yangyang Chang¹

¹School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian POCT Laboratory Dalian 116024 China mliu@dlut.edu.cn.

Chemical science

|May 10, 2024

概括

这项研究引入了一种新的CRISPR/Cas13a方法,DOES-CRISPR,它使用DNA酶显著提高诊断特异性. 这项创新克服了目标之外的问题,改善了包括病毒和基因在内的各种目标的分子检测准确性.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
遗传学是一个遗传学.

背景情况:

克里斯普尔/卡斯系统提供先进的生物传感器,但受到非目标裂变的影响,限制了实际使用.
切割RNA的DNA酶具有特定位置的切割能力,可以潜在地解决CRISPR/Cas的脱问题.

研究的目的:

开发一种名为DOES-CRISPR的新型CRISPR/Cas13a策略,利用DNA酶增强特异性.
在基于CRISPR/Cas13a的分子检测中克服非目标裂变的挑战.

主要方法:

提出了一个DNA酶操作增强CRISPR/Cas13a的特异性 (DOES-CRISPR) 策略.
设计了一个系统,其中crRNA与非目标RNA片段的部分杂交不会激活Cas13a附带活动.
针对微RNA,CYP2C19*17基因和SARS-CoV-2变体 (包括Omicron亚型) 的工程检测系统.

主要成果:

DOES-CRISPR提高了基于CRISPR/Cas13a的检测的特异性,达到43倍.
证明了对微RNA (miR-17,let-7e),CYP2C19*17基因和SARS-CoV-2变体 (玛,三角形,欧米克龙,BQ.1,XBB.1) 的单核酸解决检测.
临床评估显示,对CYP2C19*17基因型鉴定具有100%的敏感性和特异性,对Omicron和XBB.1检测具有高的诊断准确性.

结论:

DOES-CRISPR显著提高了CRISPR/Cas13a系统的特异性,解决了一个关键的局限性.
该策略显示了广泛适用于高精度检测各种核酸目标的应用.
临床验证证实了DOES-CRISPR在准确的疾病诊断和遗传分析方面的潜力.

更多相关视频

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

相关概念视频

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

Homologous Recombination

Homologous Recombination

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...