Housden Benjamin E, Perrimon Norbert
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.prot090795. doi: 10.1101/pdb.prot090795.
Although CRISPR technology allows specific genome alterations to be created with relative ease, detection of these events can be problematic. For example, CRISPR-induced double-strand breaks are often repaired imprecisely to generate unpredictable short indel mutations. Detection of these events requires the use of molecular screening techniques such as endonuclease assays, restriction profiling, or high-resolution melt analysis (HRMA). Here, we provide detailed protocols for HRMA-based mutation screening in Drosophila and analysis of the resulting data using the online tool HRMAnalyzer.
尽管CRISPR技术能够相对轻松地实现特定基因组改变，但这些事件的检测可能存在问题。例如，CRISPR诱导的双链断裂常常修复不准确，从而产生不可预测的短插入缺失突变。检测这些事件需要使用诸如核酸内切酶测定、限制性酶切图谱分析或高分辨率熔解分析（HRMA）等分子筛选技术。在此，我们提供了基于HRMA的果蝇突变筛选详细方案，以及使用在线工具HRMAnalyzer对所得数据进行分析的方法。
