Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea;
Genome Res. 2014 Jan;24(1):125-31. doi: 10.1101/gr.163394.113. Epub 2013 Nov 19.
RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F₀ and F₁ mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.
RNA 引导的内切酶（RGENs）源自原核 II 型 CRISPR-Cas 系统，可实现细胞和生物体中靶向基因组修饰。在这里，我们描述了通过注射 RGENs（Cas9 蛋白：向导 RNA 复合物或 Cas9 mRNA 加向导 RNA）到两种物种的单细胞期胚胎中，建立基因敲除小鼠和斑马鱼的方法。RGENs 可有效在新生小鼠中产生高达 93%的种系可传递突变，且毒性最小。RGEN 诱导的编码对 DNA 双链断裂修复至关重要的酶的小鼠 Prkdc 基因突变导致 F₀ 和 F₁ 代小鼠均出现免疫缺陷。我们提出，动物中的 RGEN 介导的诱变将极大地加速基因工程模型生物的创建，从而加速功能基因组研究。
