Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes

PLoS One. 2022 Jan 18;17(1):e0253351. doi: 10.1371/journal.pone.0253351. eCollection 2022.

Abstract

Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.

MeSH terms

  • Genotype*

Grants and funding

HR was funded by the Gerald Kerkut Trust. https://www.kerkut-trust.org.uk. Philippe Tardy was funded by the European Research Council Thomas Boulin was funded by the Fondation Fyssen and the European Research Council The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.