Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus

• Published in: Development Genes and Evolution Vol. 230; no. 3; pp. 257 - 264
• Main Authors: Nakayama, Ken-ichi, Ishita, Yuuki, Chihara, Takahiro, Okumura, Misako
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.05.2020; Springer Nature B.V
• Subjects: Animal Genetics and Genomics; Animals; Biomedical and Life Sciences; Cell Biology; Chemotaxis; CRISPR; CRISPR-associated endonuclease 9; CRISPR-Cas Systems; Developmental Biology; DNA; DNA sequencing; Editing; Electrophoresis, Microchip - methods; evolution; Evolutionary Biology; gene editing; Gene Editing - methods; Genetic Markers; Genome editing; Genome, Helminth; Genomes; Gonads; gRNA; heterozygosity; Heterozygote; Injection; Life Sciences; Microinjections - methods; Models, Animal; Molecular modelling; mutants; Mutation; Nematoda; Nematoda - genetics; Nematodes; Phenotype; Phenotypes; Phenotypic plasticity; Plant Genetics and Genomics; Pristionchus pacificus; progeny; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; satellites; Self-recognition; Siblings; Technical Note; Zoology; CRISPR/Cas9; Co-injection marker; Microchip electrophoresis; Pristionchus pacificus
• ISSN: 0949-944X; 3059-3239; 1432-041X; 1432-041X; 3059-3247
• DOI: 10.1007/s00427-020-00651-y

CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus , CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus . We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus .