牛受精卵 CRISPR-Cas 基因电穿孔法用于 DNA 和 RNA 靶向操作的实验方案。

Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.

机构信息

Virginia Polytechnique Institute and State University, Blacksburg, VA 24061, USA.

出版信息

STAR Protoc. 2024 Mar 15;5(1):102940. doi: 10.1016/j.xpro.2024.102940. Epub 2024 Mar 8.

DOI:10.1016/j.xpro.2024.102940

PMID:38460133

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10941008/

Abstract

The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation. The first electroporation introduces ribonucleoproteins formed by Cas9D10A with two guide RNAs to target DNA, and the second introduces the same ribonucleoprotein complex to target DNA plus Cas13a with one guide RNA to target RNAs. For complete details on the use and execution of this protocol, please refer to Nix et al..

摘要

CRISPR-Cas9 核糖核蛋白的使用彻底改变了基因组的操作。在这里，我们介绍了一种在牛受精卵中进行 DNA 和 RNA 靶向的 CRISPR-Cas 电穿孔的方案。首先，我们描述了生产和准备电穿孔用假定受精卵的步骤。第一次电穿孔将 Cas9D10A 与两条向导 RNA 形成的核糖核蛋白导入靶 DNA，第二次电穿孔将相同的核糖核蛋白复合物导入靶 DNA 加 Cas13a 和一条向导 RNA 导入靶 RNA。有关此方案的使用和执行的完整详细信息，请参阅 Nix 等人的研究。