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

• Published in: PloS one Vol. 15; no. 9; p. e0238950
• Main Authors: McDiarmid, Troy A, Au, Vinci, Moerman, Donald G, Rankin, Catharine H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.09.2020; Public Library of Science (PLoS)
• Subjects: Alleles; Animals; Antibiotics; Arrays; Automation; Biology and Life Sciences; Caenorhabditis elegans; Caenorhabditis elegans - genetics; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; CRISPR-Cas technology; Deoxyribonucleic acid; Design; DNA; Drug resistance; Efficiency; Engineering and Technology; Gene deletion; Gene Editing - methods; Gene Targeting - methods; Genes; Genetic aspects; Genetic engineering; Genome editing; Genomes; Homozygote; Life sciences; Machine vision; Methods; Mutation; Negative selection; Nematodes; Phenotype; Phenotypes; Plasmids; Proteasomes; Research and Analysis Methods; RNA, Guide, CRISPR-Cas Systems - genetics; Robustness; Screening; Toxins, Biological - genetics; Toxins, Biological - metabolism; Transgenes; Transgenic animals; Vision systems; Zoology; Canada; Vancouver British Columbia Canada
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0238950

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and 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 subunit pbs-1 and the uncharacterized gene K04F10.3 and 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.