Targeted genome editing in Caenorhabditis elegans using CRISPR/Cas9

摘要

Utilization of programmable nucleases to generate DNA lesions at precise endogenous sequences has transformed the ability to edit genomes from microbes to plants and animals. This is especially true in organisms that previously lacked the means to engineer precise genomic changes, like Caenorhabditis elegans. C. elegans is a 1mm long free-living, nonparasitic, nematode worm, which is easily cultivated in a laboratory. Its detailed genetic map and relatively compact genome (similar to 100 megabases) helped make it the first metazoan to have its entire genome sequenced. With detailed sequence information came development of numerous molecular tools to dissect gene function. Initially absent from this toolbox, however, were methods to make precise edits at chosen endogenous loci. Adapting site-specific nucleases for use in C. elegans, revolutionized studies of C. elegans biology. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and then CRISPR-associated protein 9 (Cas9) were used to target specific endogenous DNA sequences to make double-strand DNA breaks (DSBs). Precise changes could be engineered by providing repair templates targeting the DSBin trans. The ease of programming Cas9 to bind and cleave DNA sequences with few limitations has led to its widespread use in C. elegans research and sped the development of strategies to facilitate mutant recovery. Numerous innovative CRISPR/Cas9 methodologies are now primed for use in C. elegans. (C) 2017 Wiley Periodicals, Inc.