The direct repeat (DR)-GFP assay to measure the frequency of HR and the strand annealing EJ2-GFP assay to measure the frequency of MMEJ were performed as previously described30 (link). Briefly, U2OS DR-GFP or U2OS EJ2-GFP cells were transfected with 10 nM siRNA (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen). 24 h later, the cells were transfected with the pCBASceI plasmid (Addgene #26477) and plasmids, using Lipofectamine 2000 (Invitrogen). 48 h post-plasmid transfection, the cells were trypsinized and the percentage of GFP-expressing cells was analyzed using the BD FACSCalibur flow cytometer.
The Lamin A (LMNA) assay to measure the frequency of introduction of the coding sequence for mClover at the 5’ end of LMNA using the CRISPR/Cas9 was performed as previously described12 (link). Parental or 53BP1Δ U2OS cell lines were transfected with the indicated plasmids using Lipofectamine RNAiMAX (Invitrogen). 24 h later, the cells were electroporated with 2.5 µg of sgRNA plasmids and 2.5 µg of donor template using a Nucleofector (Lonza; protocol X-001). Under those condition, omission of the sgRNA gives negligible levels of mClover-positive cells12 (link). Parental or 53BP1Δ U2OS cells stably expressing CtIP-T847E mutant were transfected with an siRNA against KEAP1 and the indicated plasmids and processed as previously described12 (link).
A FACS-based gene targeting assay was developed to monitor the targeting efficiency of a Zsgreen reporter at the 3’end of Hsp90 locus. A homology repair template plasmid was generated with the coding sequence of Zsgreen and homology arms (600 base pairs) that correspond to sequence flanking the Hsp90 STOP codon. MEFs transduced with AAV i53 and control cells expressing DM were co-transfected with a repair template plasmid and a plasmid expressing Cas9 (pX330-U6-Chimeric_BB-CBh-hSpCas9) and a gRNA (5’-CGATGAGGATGCCTCGCGCA-3’). One million cells were transfected with 200ng of Cas9/gRNA plasmid and 800ng of template plasmid using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction. 16 hours later, cells were selected with puromycin (2 µg/ml) for 48 hours to enrich for Cas9-expressing cells. To determine the percentage of Zsgreen positive cells, cells were by analyzed by flow cytometry using a FACS Aria III cell sorter 8 days post-transfection.
The HISTH2BK (H2B) targeting assay was adapted from ref20 for use with Cas9 RNPs. For the targeting assay, 1.25 µg of plasmid donor was used per nucleofection.
The Lamin A (LMNA) assay to measure the frequency of introduction of the coding sequence for mClover at the 5’ end of LMNA using the CRISPR/Cas9 was performed as previously described12 (link). Parental or 53BP1Δ U2OS cell lines were transfected with the indicated plasmids using Lipofectamine RNAiMAX (Invitrogen). 24 h later, the cells were electroporated with 2.5 µg of sgRNA plasmids and 2.5 µg of donor template using a Nucleofector (Lonza; protocol X-001). Under those condition, omission of the sgRNA gives negligible levels of mClover-positive cells12 (link). Parental or 53BP1Δ U2OS cells stably expressing CtIP-T847E mutant were transfected with an siRNA against KEAP1 and the indicated plasmids and processed as previously described12 (link).
A FACS-based gene targeting assay was developed to monitor the targeting efficiency of a Zsgreen reporter at the 3’end of Hsp90 locus. A homology repair template plasmid was generated with the coding sequence of Zsgreen and homology arms (600 base pairs) that correspond to sequence flanking the Hsp90 STOP codon. MEFs transduced with AAV i53 and control cells expressing DM were co-transfected with a repair template plasmid and a plasmid expressing Cas9 (pX330-U6-Chimeric_BB-CBh-hSpCas9) and a gRNA (5’-CGATGAGGATGCCTCGCGCA-3’). One million cells were transfected with 200ng of Cas9/gRNA plasmid and 800ng of template plasmid using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction. 16 hours later, cells were selected with puromycin (2 µg/ml) for 48 hours to enrich for Cas9-expressing cells. To determine the percentage of Zsgreen positive cells, cells were by analyzed by flow cytometry using a FACS Aria III cell sorter 8 days post-transfection.
The HISTH2BK (H2B) targeting assay was adapted from ref20 for use with Cas9 RNPs. For the targeting assay, 1.25 µg of plasmid donor was used per nucleofection.
