Merkle Florian T, Neuhausser Werner M, Santos David, Valen Eivind, Gagnon James A, Maas Kristi, Sandoe Jackson, Schier Alexander F, Eggan Kevin
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Boston IVF, Waltham, MA 02451, USA.
Cell Rep. 2015 May 12;11(6):875-883. doi: 10.1016/j.celrep.2015.04.007. Epub 2015 Apr 30.
The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.
CRISPR-Cas9系统有潜力彻底改变人类多能干细胞（hPSCs）中的基因组编辑，但人们对其优势和缺陷仍知之甚少。我们系统地测试了CRISPR-Cas9在hPSCs中16个不同基因组位点介导报告基因敲入的能力。我们观察到了高效的基因靶向，但发现靶向克隆在靶向基因的两个等位基因上携带意外高频率的插入和缺失（indel）突变。这些indel由Cas9核酸酶以及Cas9-D10A单或双切口酶诱导，并且常常破坏基因功能。为克服这一问题，我们设计了在靶向等位基因上物理破坏或分离CRISPR靶位点的策略，并开发了一种生物信息学流程来识别和消除在另一个等位基因上携带有害indel的克隆。这种双管齐下的方法能够可靠地生成在靶向位点没有不需要的突变的敲入hPSC报告细胞系。
