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

PLoS One. 2020 Sep 22;15(9):e0238950. doi: 10.1371/journal.pone.0238950. eCollection 2020.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • CRISPR-Cas Systems
  • Caenorhabditis elegans / genetics
  • Caenorhabditis elegans Proteins / genetics*
  • Caenorhabditis elegans Proteins / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing / methods*
  • Gene Targeting / methods*
  • Homozygote
  • Phenotype
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Toxins, Biological / genetics*
  • Toxins, Biological / metabolism
  • Transgenes

Substances

  • Caenorhabditis elegans Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • Toxins, Biological
  • peel-1 protein, C elegans