RRC ID 57727
Author Iriki H, Kawata T, Muramoto T.
Title Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.
Journal 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.
Volume 14(10)
Pages e0224128
Published 2019-10-17
DOI 10.1371/journal.pone.0224128
PII PONE-D-19-24103
PMID 31622451
IF 2.74
Times Cited 1
Cellular slime molds G90484 G90485 G90486 G90487