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. 2018 May 9;13(5):e0197128. doi: 10.1371/journal.pone.0197128

Fig 3. Nourseothricin selection process for obtaining single-copy insertion using CRISPR/Cas9.

Fig 3

(A) Venn diagram showing the unc-4c promoter- (Punc-4c) and mig-13 promoter- (Pmig-13) expressing neurons. (B) Schematic of the protocol used to identify worms carrying the single-copy insert. The repair template vector, sgRNA vector, Cas9 vector, and three mCherry vectors are injected to N2 worms. Next day, the NTC solutions are added and the worms are allowed to grow for 7–10 days. After incubation, the healthy-looking L4/adult worms that have no mCherry signal (extrachromosomal array marker) are picked up. (C) Schematic showing the experimental design used for inserting the DA9-specific expression cassette into the defined locus. The target site for Cas9 was designed on chromosome V. Using the homology arms around the target locus, the expression cassette was inserted by homologous recombination. The resultant worms carry Cre recombinase, NTC resistance and the expression cassette, but do not carry mCherry (extrachromosomal array markers).