Housden Benjamin E, Perrimon Norbert
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):2016/9/pdb.top086843. doi: 10.1101/pdb.top086843.
The recent development of the CRISPR-Cas9 system for genome engineering has revolutionized our ability to modify the endogenous DNA sequence of many organisms, including Drosophila This system allows alteration of DNA sequences in situ with single base-pair precision and is now being used for a wide variety of applications. To use the CRISPR system effectively, various design parameters must be considered, including single guide RNA target site selection and identification of successful editing events. Here, we review recent advances in CRISPR methodology in Drosophila and introduce protocols for some of the more difficult aspects of CRISPR implementation: designing and generating CRISPR reagents and detecting indel mutations by high-resolution melt analysis.
用于基因组工程的CRISPR-Cas9系统的最新发展彻底改变了我们修改包括果蝇在内的许多生物内源DNA序列的能力。该系统允许以单碱基对精度原位改变DNA序列，目前正被用于各种各样的应用。为了有效地使用CRISPR系统，必须考虑各种设计参数，包括单向导RNA靶位点的选择和成功编辑事件的鉴定。在这里，我们回顾了果蝇CRISPR方法的最新进展，并介绍了CRISPR实施中一些较困难方面的方案：设计和生成CRISPR试剂以及通过高分辨率熔解分析检测插入缺失突变。
