Fig. 5

CRISPR-Cas9 mediated gene editing of KRAB-Zfp clusters and the effects on TE transcription in mESC. The mESC cell lines stably expressing the indicated sgRNA and inducibly expressing Cas9 were analyzed. A Verification of the efficiency and specificity of CRISPR-Cas9 mediated cleavage events in the targeted KRAB-Zfp clusters via PCR. Top panel: A Diagram of the primer design strategies. Primer pairs for directly detecting the zinc finger domains of the targeted KRAB-Zfps are indicated by F1 and R1 (the green and brown arrows) and the primers used to detect the intergenic regions of the two targeted KRAB-Zfp genes are indicated by F2 and R2 (the red and dark blue arrows). The bottom panel: The representative PCR or qPCR results for detecting the genomic DNA templates extracted before and after gene editing (−Dox and + Dox). The type of the primers is labeled on top using the arrow pairs with colors consistent with the diagram. For the specificity tests, the sgRNA labeled in red on top of the agarose gel images are the specific sgRNA designed for the KRAB-Zfp cluster of the genes tested, and the sgRNAs in black are sgRNAs designed for some other non-target clusters. The PCR products amplified from the original, non-edited control samples are indicated by pink arrows. An irrelevant DNA fragment was amplified in parallel and used as an internal control (the yellow arrow) for checking the quality and quantity of the templates used in the PCR reactions. The layout of the KRAB-Zfp genes located within the tested clusters are also shown on the top of the results and the ones with the effective PCR data are highlighted in the red boxes. For qPCR results shown at the bottom, the amplicons were located at the downstream intergenic regions of the indicated KRAB-Zfp genes. B RT-qPCR detection of the relative expression levels of the indicated classes of the repetitive DNA elements in mESC before and after gene editing (−Dox and + Dox) triggered by the sgRNAs labeled on the left. The qPCR data of the indicated TEs were normalized to the data of the Gapdh transcript, and the normalized data of −/+Dox samples of the sgRNA expressing mESC were further normalized to the data generated from the control cell line expressing the empty vector without any relevant sgRNA