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Fig. 1 | Genome Biology

Fig. 1

From: Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage

Fig. 1

A double cut HDR donor considerably increases HDR efficiency in 293 T cells after CRISPR-mediated DSB. a Schematic outline of the mCherry HDR reporter system. A lentiviral vector Lenti-EF1-Puro-sgRNA1-Wpre was used to generate reporter cell line. The red triangle indicates a sgRNA1-PAM sequence that will guide Cas9 to create DSB. 293 T cells were transduced with the lentiviral vector at a low MOI. After transduction, cells were treated with puromycin (2 ug/mL) and single-cell cloning was conducted to generate reporter cell lines with Puro-sgRNA1-Wpre target sequence (293 T reporter cells). EF1 is the promoter that drives the expression of a puromycin resistance gene. Wpre is the woodchuck hepatitis virus posttranscriptional regulatory element. After co-transfection with promoterless mCherry donor and two plasmids encoding Cas9 and sgRNA1, the 293 T reporter cells use the donor to repair DSB by HDR pathway leading to the integration and expression of mCherry. b Design of promoterless mCherry HDR donors. pD-mCherry is a conventional circular HDR donor and pD-mCherry-sg is a double cut HDR donor in which the Puro-mCherry-Wpre cassette is flanked by two sgRNA1 recognition sequences. Puro (663 bp) and Wpre (592 bp) serve as left and right HA, respectively. To simplify naming scheme, the length of Puro and Wpre are unified as 600 bp and the tag HA600-600 bp indicates their HA length. c FACS analysis of 293 T reporter cells one week after co-transfection of Cas9 and conventional vs. double cut pD-mCherry donors, with or without sgRNA1. The portions of mCherry+ cells represent the HDR-mediated knockin efficiencies. d HDR efficiency by two different donors. n = 3; error bars represent S.E.M. Significance was calculated using the Student’s paired t-test: **P ≤ 0.01

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