dictyNews

Electronic Edition

Volume 45, number 27

October 25, 2019



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Abstracts

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Generation of deletions and precise point mutations in Dictyostelium 

discoideum using the CRISPR nickase



Hoshie Iriki, Takefumi Kawata, Tetsuya Muramoto





PLOS One, 14 e0224128



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.





submitted by:  Tetsuya Muramoto [[log in to unmask]]

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[End dictyNews, volume 45, number 27]