To construct sgRNA-expressing plasmids, complementary oligos representing the target sequences were annealed and cloned into pFYF1320 (Addgene #47511). To construct pegRNA-expressing plasmids, complementary oligos representing the target sequences, an sgRNA scaffold, and 3' extensions were annealed and cloned into the pU6-pegRNA-GG-acceptor (Addgene #132777). The oligos are listed in Table S3.
MEFs were transfected with 750 ng of the PE2-encoding plasmid (Addgene #132775), 250 ng of the pegRNA-encoding plasmid (Addgene #132777), 100 ng of the sgRNAencoding plasmid (Addgene #47511), and 50 ng of a plasmid expressing a puromycin resistance gene. Transfected cells were then cultured for 2 days with puromycin (3 µg/ml).
pCMV-PE2 was a gift from David Liu (Addgene plasmid #132775) [22] ; pFYF1320 EGFP Site#1 was a gift from Keith Joung (Addgene plasmid #47511) [23] ; and the pU6-pegRNA-GG-acceptor was a gift from David Liu (Addgene plasmid #132777) [22] .
MEFs were transfected with 750 ng of the PE2-encoding plasmid (Addgene #132775), 250 ng of the pegRNA-encoding plasmid (Addgene #132777), 100 ng of the sgRNAencoding plasmid (Addgene #47511), and 50 ng of a plasmid expressing a puromycin resistance gene. Transfected cells were then cultured for 2 days with puromycin (3 µg/ml).
pCMV-PE2 was a gift from David Liu (Addgene plasmid #132775) [22] ; pFYF1320 EGFP Site#1 was a gift from Keith Joung (Addgene plasmid #47511) [23] ; and the pU6-pegRNA-GG-acceptor was a gift from David Liu (Addgene plasmid #132777) [22] .