| RRC ID |
57727
|
| 著者 |
Iriki H, Kawata T, Muramoto T.
|
| タイトル |
Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.
|
| ジャーナル |
PLoS One
|
| Abstract |
The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10-30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum.
|
| 巻・号 |
14(10)
|
| ページ |
e0224128
|
| 公開日 |
2019-10-17
|
| DOI |
10.1371/journal.pone.0224128
|
| PII |
PONE-D-19-24103
|
| PMID |
31622451
|
| PMC |
PMC6797129
|
| MeSH |
Base Sequence
CRISPR-Cas Systems / genetics*
Deoxyribonuclease I / genetics
Deoxyribonuclease I / metabolism
Dictyostelium / genetics*
Gene Editing / methods
Point Mutation
RNA, Guide, Kinetoplastida / genetics
RNA, Guide, Kinetoplastida / metabolism
|
| IF |
2.74
|
| 引用数 |
1
|
| リソース情報 |
| 細胞性粘菌 |
G90484
G90485
G90486
G90487 |
