Ruis Brian L, Bielinsky Anja K, Hendrickson Eric A
Department of Medicine, University of Virginia, Charlottesville, VA, USA.
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA.
Exp Mol Med. 2025 Jul;57(7):1409-1418. doi: 10.1038/s12276-025-01442-z. Epub 2025 Jul 31.
Gene editing is the intentional modification of a genetic locus in a living cell and is used for two general applications of great importance and wide interest. One is the inactivation of genes ('knockouts'), a process utilized to delineate the loss-of-function phenotype(s) of a particular gene. The second application ('knock-ins') is essentially the process of gene therapy, which predominately involves correcting a pre-existing mutated allele(s) of a gene back to wild-type to ameliorate some pathological phenotype associated with the mutation. Importantly, although these applications are conceptually exact reciprocal opposites of one another, they are achieved via mechanistically different pathways. In the case of knockouts, breakage (usually in the form of double-stranded breaks) of the chromosomal DNA at the site of targeting is used to engage a repair process (nonhomologous end joining) that is error prone. The ensuing repair frequently results in insertions/deletions at the cleavage site, which, in turn, results in out-of-frame mutations and, hence, a knockout of the gene in question. In the case of knock-ins, breakage (again, usually in the form of double-stranded breaks) of the DNA is used to engage a repair process (homology-dependent repair/recombination) in which homologous sequences between an incoming donor DNA (containing new genetic information) and the chromosomal DNA are exchanged. Although homology-directed repair was known to predominate in bacteria and lower eukaryotes, the competing process of nonhomologous end joining predominates in higher eukaryotes and was presumed to prevent the use of knock-in gene editing in human somatic cells in culture. A series of molecular and technical advances disproved this notion but still resulted in a process that was cumbersome, labor intensive, highly inefficient and slow. In 2013, however, a new RNA-programmable nuclease, CRISPR-Cas9 was described that has revolutionized the field and made gene editing accessible to anyone with even a rudimentary knowledge of molecular biology. Thus, gene editing in a wide variety of model organisms, as well as human somatic cells in culture, has become not only extremely feasible but also extremely facile, and it harbingers a golden age for directed mutagenesis, directed evolution and improvements in gene therapy.
基因编辑是对活细胞中基因位点进行的有意修饰，用于两个具有重大意义且广受关注的一般应用。一是基因失活（“敲除”），该过程用于描绘特定基因的功能丧失表型。第二个应用（“敲入”）本质上是基因治疗过程，主要涉及将基因预先存在的突变等位基因校正回野生型，以改善与该突变相关的某些病理表型。重要的是，尽管这些应用在概念上是彼此完全相反的，但它们是通过机制不同的途径实现的。在敲除的情况下，靶向位点处的染色体DNA断裂（通常以双链断裂的形式）用于启动一个易错的修复过程（非同源末端连接）。随后的修复经常导致切割位点处的插入/缺失，进而导致移码突变，从而使相关基因敲除。在敲入的情况下，DNA断裂（同样，通常以双链断裂的形式）用于启动一个修复过程（同源依赖性修复/重组），其中导入的供体DNA（包含新的遗传信息）与染色体DNA之间的同源序列会发生交换。尽管已知同源定向修复在细菌和低等真核生物中占主导地位，但非同源末端连接的竞争过程在高等真核生物中占主导地位，并且据推测这会阻止在培养的人类体细胞中使用敲入基因编辑。一系列分子和技术进步推翻了这一观念，但仍然导致了一个繁琐、劳动密集、效率极低且缓慢的过程。然而，在2013年，一种新的RNA可编程核酸酶CRISPR-Cas9被报道，它彻底改变了该领域，使任何甚至仅具有基本分子生物学知识的人都能进行基因编辑。因此，在各种模式生物以及培养的人类体细胞中进行基因编辑不仅变得极其可行，而且极其简便，它预示着定向诱变、定向进化和基因治疗改进的黄金时代。
