Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans

摘要

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleo-protein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4 days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.