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. 2014 May 30;197(4):1069–1080. doi: 10.1534/genetics.114.166389

Figure 4.

Figure 4

A blasticidin-resistance marker to select pie-1 knockout mutants. (A) Schematic of the Cas9/sgRNA target sequence and an HR donor plasmid in which a heterologous blasticidin-resistance (BSD) gene replaces a region of pie-1 and is flanked by 1-kb homology arms. The BSD gene is under the control of the rpl-28 promoter (568 bp) and 3′-UTR (568 bp). (B) Schematic of the blasticidin selection strategy to precisely delete the pie-1 gene. pie-1a sgRNA was co-injected with the Cas9 expression vector, the rol-6 transformation marker, the pie-1∆::BSD donor construct, and the pCCM416::Pmyo-2::avr-15(+) counterselection vector. The indicated number of F1 rollers was transferred to the plates containing 2 ng/ml ivermectin to select against the extrachromosomal array and 100 μg/ml blasticidin to identify BSD knock-in lines. We identified two plates with resistant, fertile adults among 14 plates, 3–4 days after transferring animals.