相关实验视频

Updated: Jun 25, 2025

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

Published on: February 17, 2023

混合Cas12a变种与放松的PAM要求扩大基因组编辑兼容性

Zhenyu Liu¹, Huayi Liu¹, Chaoqun Huang¹

¹Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

ACS synthetic biology

|May 31, 2024

概括

蛋白质工程创造了一个新的Cas12a核酶 (ehCas12a),可以编辑更广泛的DNA序列. 一种变种,ehCas12a RRVR,针对以前无法访问的PAM网站进行潜在的基因疗法应用.

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:

Cas12a核酶是基因组编辑的重要工具,但受到其特定的PAM识别序列的限制.
正在探索蛋白质工程策略,以克服这些局限性,并扩大Cas12a系统的实用性.

研究的目的:

为了设计Cas12a变体,改变和扩展Protospacer相邻动机 (PAM) 识别.
开发基于Cas12a的新型工具,用于基因组编辑和基因编辑,具有更广泛的准能力.

主要方法:

设计和选了990个使用域混合的合成混合Cas12a嵌合体.
构建了一个高效的混合Cas12a (ehCas12a) 具有广泛的PAM识别 (5'-TYYN-3').
开发了一种ehCas12a RRVR变种,具有扩展的PAM偏好 (5'-TNYN,TWRV-3') 并测试了DNase失活的版本 (dehCas12a RRVR-BE) 用于体内应用.

主要成果:

确定了一种高效的Cas12a (ehCas12a) 混合物,可以识别广泛的5'-TYYN-3' PAM范围.
设计了ehCas12a RRVR变体,以识别具有挑战性的2* A/G PAM.
通过 dehCas12a RRVR-BE 基编辑器在体内证明了非正规PAM和与疾病相关的位置的向.

结论:

通过模块化设计和域混合,蛋白质工程可以显著提高Cas蛋白的功能.
开发的ehCas12a及其变体扩大了Cas12a系统的基因组编辑和基因编辑的目标范围.
这些工程核酶有望用于治疗应用,因为它们可以在以前无法访问的基因组部位进行精确的编辑.

关键词:

这就是Cas12a.在 PAM 认可中使用 PAM 识别.基础编辑基础编辑域名混合是指域名的混合.合成生物学合成生物学

更多相关视频

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Published on: May 10, 2020

相关概念视频

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...

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...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

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...

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...