Journal: RNA

Article Title: A piggyBac-based toolkit for inducible genome editing in mammalian cells

doi: 10.1261/rna.068932.118

Figure Lengend Snippet: Inducible protein-coding gene knockdown with CRISPR-Bac. See Supplemental Table S2 for details on replicates and experimental design for each figure panel. ( A ) Western blot of CRISPR-Bac assay in which three separate sgRNAs targeting Ezh2 , a pool of all three sgRNAs, or a nontargeting sgRNA control (“No sgRNA”) were cotransfected along with the inducible Cas9-expressing piggyBac cargo into E14 mouse ESCs. Western blots to EZH2 and TBP were performed on protein extracted from stably selected cells, after 4 d of Cas9 induction with doxycycline. Values underneath blots represent knockdown of EZH2 relative to no sgRNA controls and normalized for loading with TBP protein levels. ( B ) Biological replicates of experiment in A for sgRNA pool. “Pool rep #1” is the same sample as in A . ( C ) Representative IF image showing EZH2 knockdown in nontargeting sgRNA control (“no sgRNA”) or pooled sgRNA cells from replicate #2 in B . Scale bar, 10 µm. ( D , E ) Western blot and IF to EZH2 in transient transfection experiments with pX330.

Article Snippet: Protein knockdown sgRNAs were designed using Desktop Genetics, and all other sgRNAs were designed using the CRISPOR program or taken from published sources ( Supplemental Table S1 ; ).

Techniques: Knockdown, CRISPR, Western Blot, BAC Assay, Control, Expressing, Stable Transfection, Transfection

Journal: RNA

Article Title: A piggyBac-based toolkit for inducible genome editing in mammalian cells

doi: 10.1261/rna.068932.118

Figure Lengend Snippet: The CRISPR-Bac system functions in human cell lines. See Supplemental Table S2 for details on replicates and experimental design for each figure panel. ( A ) Western blots to EZH2 and ERK2 loading control from two replicate CRISPR-Bac experiments in SUM-159 cells. “No Cas9” refers to measurements taken from dCas9-VP160/rtta- Il1RN sgRNA expressing SUM-159 cells that were cultured in parallel to those expressing Cas9 and the EZH2 sgRNA pool. Values underneath blots represent knockdown of EZH2 relative to No Cas9 control and normalized for loading with ERK2 protein levels. ( B ) Representative IF images showing EZH2 knockdown from “No Cas9” and “EZH2 pool, rep #1” SUM-159 cells in A . Image #1 shows a cell with partial knockdown next to a cell with full knockdown, and image #2 shows two cells with full knockdown. Scale bar, 10 µm. ( C ) qPCR results showing transcriptional activation of Il1RN in SUM-159 cells. In panel C , “No dCas9” refers to measurements taken from Cas9/rtta- EZH2 sgRNA expressing SUM-159 cells that were cultured in parallel to those expressing dCas9 and the Il1RN sgRNAs. Data from the nontargeting sgRNA control (No sgRNA) and sgRNA-expressing cells are plotted relative to the average of the signal in the No sgRNA control cells. Individual qPCR data points are shown in box-and-whisker format. (***) P < 0.001 from a two-sided t -test between no sgRNA and sgRNA-expressing cells.

Article Snippet: Protein knockdown sgRNAs were designed using Desktop Genetics, and all other sgRNAs were designed using the CRISPOR program or taken from published sources ( Supplemental Table S1 ; ).

Techniques: CRISPR, Western Blot, Control, Expressing, Cell Culture, Knockdown, Activation Assay, Whisker Assay