Hubrecht Institute and UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine at Utrecht University, Yalelaan 108, 3583 CM Utrecht, the Netherlands.
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Stem Cell Reports. 2015 Nov 10;5(5):908-917. doi: 10.1016/j.stemcr.2015.09.022. Epub 2015 Oct 29.
We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%-4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.
我们提出了自我克隆 CRISPR/Cas9（scCRISPR）技术，该技术通过绕过为每个靶标位点克隆特定 sgRNA 或同源敲入构建体的需要，允许在数小时内实现 CRISPR/Cas9 介导的基因组突变和特定位点的敲入转基因的创建。我们将自我切割的回文 sgRNA 质粒和短的双链 DNA 序列编码所需的靶标特异性 sgRNA 导入靶细胞，使它们能够通过同源重组产生靶标特异性 sgRNA 质粒。scCRISPR 能够在大约六分之一的基于质粒的 sgRNA 构建成本下，有效地在基因敲除（约 88%的突变率），每个靶位点的准备时间仅为 2 小时。此外，我们还证明了在小鼠和人类胚胎干细胞中，通过短同源臂的 PCR 添加，高效地进行 GFP 转基因的特定位点敲入，而无需任何质粒克隆或基因组整合选择盒（2%-4%的敲入率）。scCRISPR 大大降低了小鼠和人类转基因的门槛。
