利用组装的 CRISPR-Cas9 核糖核蛋白复合物进行基因组编辑。
Genome editing in using assembled CRISPR-Cas9 ribonucleoprotein complexes.
机构信息
Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.
Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India; DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.
出版信息
STAR Protoc. 2022 Aug 19;3(3):101629. doi: 10.1016/j.xpro.2022.101629. eCollection 2022 Sep 16.
The plasmid-free CRISPR-Cas9-based genome editing in fungi is a precise and time-saving approach. Here, we present a detailed protocol for genetic manipulation in , which includes design and synthesis of sgRNA, high-quality protoplast preparation, and PEG-mediated protoplast transformation of linear donor DNA along with synthesized RNP complex composed of sgRNA and host-specific Cas9. This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment. For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021).
在真菌中使用无质粒细胞 CRISPR-Cas9 进行基因组编辑是一种精确且省时的方法。在这里，我们提供了一个在 中进行遗传操作的详细方案，包括 sgRNA 的设计和合成、高质量原生质体制备，以及 PEG 介导的线性供体 DNA 的原生质体转化，以及由 sgRNA 和宿主特异性 Cas9 组成的合成 RNP 复合物。对于有兴趣对基因进行功能分析的研究人员来说，这项技术提高了实验的可重复性和可复制性，因此非常有益。有关此方案的使用和执行的完整详细信息，请参见 Randhawa 等人（2021）。
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