Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

doi: 10.1073/pnas.1711979114

Figure Lengend Snippet: PCR fragments with short homology arms are efficient donors to create GFP knockins in HEK293T cells. ( A ) Diagrams showing PCR donors for GFP insertion at the Lamin A/C and RAB11A loci. Locus, gray; GFP, green; homology arms, blue; and DSB, vertical line. GFP was inserted at the DSB in Lamin A/C and 11 bp upstream of the DSB in RAB11A . ( B ) Graphs showing percentage of GFP + cells obtained with PCR donors with homology arms of the indicated lengths (33/33 refers to a right homology arm and a left homology arm, each 33 bp long). Insert size in all cases was 714 bp. Each bar represents the average insertion efficiency from two or more independent experiments ( SI Appendix , Table S1 ). Error bars represent the ±SD. PCR fragments were nucleofected in HEK293T cells at the concentration indicated and counted by flow cytometer 3 d later. For this and all other figures, SI Appendix , Table S1 provides details. ( C ) Graphs showing percentage of GFP + cells obtained with PCR or plasmid donors with homology arms of the indicated lengths. Insert size in all cases was 714 bp. Each bar represents the average insertion efficiency from two or more independent experiments ( SI Appendix , Table S1 ). Error bars represent the ±SD. PCR fragments were nucleofected in HEK293T cells at the concentration indicated and cells were counted by flow cytometer 3 d later. ( D ) Confocal images of cells 3 d after nucleofection. GFP, green; DNA, blue. The GFP subcellular localizations are as expected for in-frame translational fusions.

Article Snippet: Just before nucleofection, PBS was replaced with 80 µL of Nucleofection kit V (Lonza).

Techniques: Concentration Assay, Flow Cytometry, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

doi: 10.1073/pnas.1711979114

Figure Lengend Snippet: Polarity of ssODNs affects incorporation of distal edits. ( A ) Schematics showing possible pairing interactions between resected locus (gray) and ssODNs (light or dark blue for sense and antisense ssODN, respectively, arrows indicate 3′ ends) coding for a distal insert (green). Sequences between the DSB and insert were recoded to help integration of the distal insert and prevent cutting of edited locus by Cas9. ( B ) Normalized efficiency of sense versus antisense ssODNs calculated as in ( SI Appendix , Table S1 provides detailed results). Distance from the DSB, locus, and guide RNA polarity are indicated Below each experiment. ssODN polarity has little effect on editing efficiency for proximal edits, but has a larger effect for distal edits. The favored polarity changes, depending on whether the distal edit is positioned to the left or right of the DSB. Note that the favored ssODN polarity does not correlate with crRNA polarity (for example, first two columns in the graph show crRNA1776 and crRNA1777, which cut at the same position but have opposite polarity). Experiments involving the PYM1 locus were done on HEK293T that were cloned out and genotyped by PCR genotyping (size shift) for 3×Flag insertion . All other experiments were performed on HEK233T (GFP1–10) cells that were directly scored for GFP + by flow cytometer or microscopy 3 d after nucleofection. Numbers Above each column indicate the overall percentage of edits. Note that overall frequency decreases with increasing distance from the DSB (also see SI Appendix , Fig. S6 ).

Article Snippet: Just before nucleofection, PBS was replaced with 80 µL of Nucleofection kit V (Lonza).

Techniques: Clone Assay, Flow Cytometry, Microscopy