Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of Cas9 fused to the HUH endonuclease PCV with a covalently attached ssODN. ( b ) SDS-PAGE of Cas9 variants reacted with an Alexa 488 fluorescently labelled ssDNA containing the PCV recognition sequence. The top panel is the coomassie stained gel, and the bottom panel is the identical fluorescently imaged gel. PCV is fused to either the carboxyl (Cas9-PCV) or amino (PCV-Cas9) terminus of Cas9. Cas9-PCV(Y96F) represents catalytically inactive PCV(Y96F) fused to Cas9. (C) SDS-PAGE gel shift assay of Cas9 reacting with two ssDNA templates containing the PCV recognition sequence of differing lengths in a 1:1 ssDNA:Cas9 molar ratio.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: SDS Page, Sequencing, Staining, Electrophoretic Mobility Shift Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of split luciferase insertion. The C-terminus of NanoLuc nanoluciferase (HiBiT) is encoded on the 200bp ssODN along with the 5’ PCV recognition sequence and targeted to the 3’ end of GAPDH . ( b ) Assaying luminescence using different Cas9 variants when inserting HiBiT into GAPDH in HEK-293T cells. PCV is fused to either the amino (PCV-Cas9) or carboxyl (Cas9-PCV) terminus of Cas9. Transfections were performed with ssODN lacking the PCV recognition sequence (PCV- ssODN) or ssODN containing the PCV recognition sequence (PCV+ ssODN). Units are displayed in relative light units (RLU) normalized to Cas9. ( c ) The calculated fold change from (b) between the PCV-ssODN and PCV+ ssODN is shown for each variant. ( d ) Targeting the GAPDH locus in U2-OS cells. ( e ) Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. ( f ) Fold change in RLU compared to Cas9 when varying the amount of RNP (equimolar ssODN). All graphs represent data from one of multiple independent experiments exhibiting similar results. Data are shown as mean +/− SD (n=3). Significance calculated using 2-tailed Student’s t-test: ** P < 0.01, *** P < 0.001, ns = no significance (P >0.05).

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Luciferase, Sequencing, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) HEK-293T cells stably expressing a mutant mCherry-GFP reporter are edited by HDR through a frameshift correction, restoring mCherry activity. ( b ) Representative microscopy images of fluorescent reporter editing. ( c ) The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using an ssODN containing the PCV recognition sequence ( d ) RNP transfections at 3 pmol in the presence or absence of ssODN. Data are shown as mean +/− SD (n=3). For (c) and (d), the statistical significance of %mCherry positive cells between PCV-fusions of Cas9 and Cas9 alone was <0.001, calculated using 2-tailed Student’s t-test.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay, Microscopy, Flow Cytometry, Sequencing, Transfection