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. 2019 Aug;25(8):1047–1058. doi: 10.1261/rna.068932.118

FIGURE 1.

FIGURE 1.

Experimental pipeline used for CRISPR-Bac. In a CRISPR-Bac experiment, a Cas9-expressing piggyBac cargo vector (or dCas9 variant) is cotransfected with a sgRNA- and rtTA-expressing piggyBac cargo vector and with a piggyBac transposase plasmid. Growth in Hygromycin B and G418 for 7 to 12 d selects for a population of cells that stably express an sgRNA of interest, and inducibly express a Cas9 or dCas9 variant. TR, piggyBac inverted terminal repeat. HS4, chicken βglobin insulator element. TRE, tetracycline responsive element (i.e., doxycycline inducible promoter). hCas9, dCas-VP160, dCas-KRAB from Cheng et al. (2013), Cong et al. (2013), and Kearns et al. (2014). EF1, EF1α promoter. HygroR, Hygromycin B resistance gene. SV40pA, SV40 polyadenylation signal. hU6-chimeric sgRNA from Cong et al. (2013) with BsmbI sites replacing BbsI sites. hUbiC-rtTA3-IRES-G418 cassette was from pSLIK-Neo (Shin et al. 2006).