Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans.

Abstract

Streamlined, selection-based CRISPR knock-in protocols for C. elegans were first introduced six years ago (Dickinson et al. 2015; Schwartz and Jorgensen 2016). Though these selection-based approaches are powerful, one drawback has been the requirement to inject large numbers of P0 worms (~30-60 per gene target). We have found that a combination of high-purity DNA and a lower concentration of Cas9/sgRNA plasmid dramatically improves efficiency, often resulting in multiple independent CRISPR knock-ins via as few as 10 injected worms, comparable to the efficiency of melted dsDNA templates and purified Cas9 protein (Dokshin et al. 2018; Ghanta and Mello 2020).