Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase


Autoři: Hoshie Iriki aff001;  Takefumi Kawata aff001;  Tetsuya Muramoto aff001
Působiště autorů: Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba, Japan aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224128

Souhrn

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.

Klíčová slova:

Cloning – CRISPR – Deletion mutation – Dictyostelium discoideum – Point mutation – Polymerase chain reaction – Vector cloning – Oligonucleotides


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2019 Číslo 10