Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
STAR Protoc. 2022 Aug 19;3(3):101551. doi: 10.1016/j.xpro.2022.101551. eCollection 2022 Sep 16.
In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Panday et al. (2021).
在本方案中，我们使用 CRISPR/Cas9 对目的基因的整个编码区进行大片段缺失，从而产生半合子细胞系。接下来，我们在完整的野生型等位基因内系统地构建精确的框内缺失，从而便于研究多结构域蛋白。本文描述的优化方案使我们能够快速筛选有效的 sgRNA 对，并在小鼠胚胎干细胞中高频构建框内缺失或移码突变。有关本方案使用和实施的完整详细信息，请参见 Panday 等人（2021 年）。
