MOESM1 of Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells
Creators: name: Hu, Zheng
name: Shi, Zhaoying
name: Guo, Xiaogang
name: Jiang, Baishan
name: Wang, Guo
name: Luo, Dixian
name: Chen, Yonglong
name: Zhu, Yuan-Shan
Publisher: name: figshare
Year: 2018
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Evolutionary Biology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1. A schematic illustration for constructing a Cas9, eGFP and gRNA co-expression vector. Figure S2. Determination of insertion repair efficiency at AAVS1 locus by DNA sequencing. Panel A shows a representative DNA sequencing of a TA clone without a mutation induced by Cas9 and insertion repair at the AAVS1 locus. The Cas9 targeted site of the AAVS1 locus is underlined. Panel B shows a representative DNA sequencing that confirms the incorporation of an ssODN-harbored EcoRI site at the targeted position of the AAVS1 locus. The EcoRI site is underlined. Panel C shows a representative DNA sequencing of a TA clone with NHEJ, but without insertion repair at the AAVS1 locus. The mutation sequences induced by Cas9 are underlined. Figure S3. The generation and validation of a GFP-silent mutation lentivirus vector and MCF-7/GFP-Mut cells. Panel A shows part of the GFP ORF with a premature termination codon, tGA, through a replacement of two nucleotides by GA in the sequence (GFP-Mut). Panel B shows a representative fluorescence image of 293T cells used for the package of lentivirus by co-transfecting the pSIN-EF1-GFP-Mut-Puromycin (left) or GFP-Wild type control (right) lentivirus vector together with auxiliary plasmids pSPAX2 and pMD2.G. Forty-eight hours after transfection, the supernatants were collected and the transfected 293T cells were examined by fluorescence microscope (5×). Fluorescence signal is undetectable in 293T cells transfected with GFP-Mut vector (left). Panel C shows that the replacement of two nucleotides, ac, in the wild-type, by GA leads to a formation of a termination codon tGA and a change in the PAM sequence. Panel D shows a representative gel image of T7E1 cleavage assay of disruption efficiency in MCF-7/GFP-Mut cells transfected by Cas9 and GFP-Mut sgRNA. Figure S4. Schematic diagrams for DNA sequencing of single cell-derived clones. Single GFP+ cell-derived clones were used to analyze homology-directed repair (HDR) and to evaluate the mutation-corrected rate. Genomic DNA from cell clones was PCR amplified, and the PCR products were sequenced directly. Representative DNA sequencing of cell clones are: Panel A – control HCT-116 cells; Panel B – gene mutation corrected cells; Panel C – cells with HDR but without mutation correction; and Panel D – cells without HDR. Table S1. PCR primers and oligonucleotides used for cloning sgRNA expression vector, HDR-mediated repair and Cas9 target sites.