CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.
Keywords: Pristionchus pacificus; CRISPR/Cas9; Co-injection marker; genome editing; microinjection.
© 2021 Japanese Society of Developmental Biologists.