Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adaptedpeel-1negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering inCaenorhabditis elegansand observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunitpbs-1and the uncharacterized geneK04F10.3and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.