Ruthenium Red 钌红CAS： 11103-72-

张峰手稿：基因工程技术之CRISPR-Cas9

transfection. Maintain the cells for another 72 hto allow suffi cient time for HR mediated by CRISPR-Cas9 systemand the template. 3.5 Verifi cation and Quantifi cation of HR Effi ciency To verify the homologous recombination between the HR

Assisted large fragment insertion by Red/ET-recombination (ALFIRE)―an alternative and enhanced method for large fragment recombineering

/RecT from the Rac phage or Redα/Redβ from the λ phage, to assist in the cloning or subcloning of fragments of DNA into vectors without the need of restriction enzyme sites or ligases (7,8). A limitation of the original homologous recombination technique

CRISPR-Cas9 全程实操教程：从 gRNA 设计到挑单克隆全程指导

基本原理CRISPR-Cas9 是一种细菌获得性免疫，用于降解入侵的外源 DNA。CRISPR RNA (crRNA) 与转录激活 crRNA (Trans-activating crRNA， tracrRNA) 退火形成的复合物能特异性识别与其互补的基因组序列，引导 Cas9 核酸内切酶在目的片段生成 DNA 双链断裂，如下图所示。通过基因工程手段对 crRNA 和 tracrRNA 进行改造，将其连接在一起得到 sgRNA (single guide RNA)。通过将表达 sgRNA