CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium.

ABSTRACT: CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the indi

SUBMITTER: Sekine R 

PROVIDER: S-EPMC5981456 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature