Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA;
Department of Biology, University of Virginia, Charlottesville, Virginia 22904, USA
Cold Spring Harb Protoc. 2022 Mar 1;2022(3):pdb.prot106971. doi: 10.1101/pdb.prot106971.
CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR-Cas9 can be used to create either mosaic loss-of-function embryos that display F generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR-Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.
CRISPR-Cas9 基因编辑技术被广泛用于在二倍体青蛙中创建靶向功能丧失突变。在这里，我们描述了一种简单的基因编辑方法，通过将 Cas9 蛋白或 mRNA 与针对特定 DNA 序列的合成向导 RNA（sgRNA）一起微注射到早期胚胎中。Cas9 催化的双链断裂会经历易错修复，导致产生短插入和/或缺失。因此，仔细选择靶位点可以导致突变，从而损害蛋白质产物的正常功能。CRISPR-Cas9 可用于创建显示 F 代表型的嵌合功能丧失胚胎或用于下游分析的突变系。除了描述如何使用 CRISPR-Cas9 进行基因诱变外，我们还讨论了一种简单的方法来确定诱变效率、可能出现的一些潜在问题以及可能的解决方案。这里描述的方案应该适用于其他两栖动物，并且原则上适用于许多其他生物体。
