使用MACHETE (用工程合元件分子改变染色体) 进行工程大基底大小的基因组删除
Francisco M Barriga1, Scott W Lowe2
1Systems Oncology Program, Vall d'Hebron Institute of Oncology (VHIO), Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain. fbarriga@vhio.net.
Nature protocols
|February 7, 2024
概括
我们开发了MACHETE,这是一种CRISPR-Cas9系统,可以高效地进行高达45Mb的大型基因组删除工程. 这种方法选择了删除事件,极大地丰富了所需的细胞变化.
科学领域:
- 基因组学就是基因组学.
- 分子生物学分子生物学
- 基因编辑 基因编辑
背景情况:
- 特定站点的核酶有助于基因组区域的消除.
- 由于效率下降和罕见的目标事件,大型基因组删除具有挑战性.
研究的目的:
- 开发一种用于高效工程的大型基因组删除的新系统.
- 克服当前方法在实现大规模基因编辑方面的局限性.
主要方法:
- 开发了一种基于CRISPR-Cas9的两阶段系统MACHETE (染色体的分子改变与工程合元件).
- 涉及插入一个选择磁带,然后引入单导向RNA来工程删除.
- 利用负选择对已经失去目标位置的细胞进行丰富.
主要成果:
- 马切特能够进行最多45 Mb的删除工程.
- 该系统通常在4-6周内产生所需的变化.
- 成功地用于快速创建等位基序列以映射基位活动.
结论:
- 马切特显著提高了工程大基因组删除的效率.
- 该协议提供了一个强大的方法来创建大数据库大小的删除.
- 潜在的应用包括癌症遗传学,转录调节和基因组架构研究.
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