Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus. (16th December 2021)

• Record Type: Journal Article
• Authors: Hiraga, Hirokuni; Ishita, Yuuki; Chihara, Takahiro; Okumura, Misako
• Other Names: Ogino H. guestEditor.; Kamei Y. guestEditor.; Hayashi T. guestEditor.; Sakamoto J. guestEditor.; Suzuki M. guestEditor.; Igawa T. guestEditor.
• Is Part Of: Development growth and differentiation. Volume 63:Number 9(2021)
• Page Start: 488
• Page End: 500
• Publication Date: 2021-12-16
• Subjects: Co‐injection marker -- CRISPR/Cas9 -- genome editing -- microinjection -- Pristionchus pacificus; Embryology -- Periodicals; Developmental biology -- Periodicals; Growth -- Periodicals; 574.3
• DOI: 10.1111/dgd.12761
• Languages: English
• ISSNs: 0012-1592

Abstract

Abstract: 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. Abstract : In the nematode Pristionchus pacificus, CRISPR/Cas9‐mediated mutations can be induced by microinjection of Cas9 protein and guide RNA into the gonads. We describe the co‐injection strategy using two types of markers to efficiently find genome‐edited animals. Microinjection, which is the most difficult and important step in mutagenesis, has also been described, including tips for success.