| RRC ID |
52782
|
| Author |
Sekine R, Kawata T, Muramoto T.
|
| Title |
CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium.
|
| Journal |
Sci Rep
|
| 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 individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.
|
| Volume |
8(1)
|
| Pages |
8471
|
| Published |
2018-5-31
|
| DOI |
10.1038/s41598-018-26756-z
|
| PII |
10.1038/s41598-018-26756-z
|
| PMID |
29855514
|
| PMC |
PMC5981456
|
| MeSH |
Base Sequence
CRISPR-Cas Systems / genetics*
Dictyostelium / genetics*
Dictyostelium / metabolism
Gene Editing / methods*
Gene Frequency
Genetic Vectors / genetics
Genetic Vectors / metabolism
Genome, Protozoan*
Mutagenesis
Phosphatidylinositol 3-Kinases / genetics
Protozoan Proteins / genetics*
RNA, Guide, Kinetoplastida / chemistry
RNA, Guide, Kinetoplastida / genetics
RNA, Transfer / metabolism
|
| IF |
3.998
|
| Times Cited |
16
|
| Resource |
| Cellular slime molds |
G90426
G90427
G90428
G90429
G90455
G90456
G90457
G90458
G90459 |
