Reisch Christopher R, Prather Kristala L J
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida.
Curr Protoc Mol Biol. 2017 Jan 5;117:31.8.1-31.8.20. doi: 10.1002/cpmb.29.
The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc.
利用位点特异性DNA核酸内切酶系统CRISPR/Cas9的基因组编辑系统的发现和发展，从根本上改变了许多生物体中基因组编辑的便捷性和速度。在真核生物中，CRISPR/Cas9系统利用“向导”RNA使Cas9核酸酶在特定的基因组位点产生双链断裂，该断裂由非同源末端连接（NHEJ）修复酶修复，在此过程中常常产生随机突变。通过在体内提供带有所需突变的DNA模板，也可以产生目标基因组的特定改变，该模板通过同源定向修复被整合。然而，大肠杆菌缺乏强大的双链断裂修复系统。因此，与真核生物不同，用Cas9靶向大肠杆菌染色体DNA会导致细胞死亡。然而，Cas9介导的细菌杀伤可用于在混合群体中筛选具有特定基因型的细胞。结合已充分描述的用于大肠杆菌重组的λ-Red系统，我们创建了一个高效的无标记和无疤痕基因组编辑系统。© 2017约翰威立父子公司
