Journal: ACS Central Science

Article Title: Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

doi: 10.1021/acscentsci.0c00129

Figure Lengend Snippet: Demonstration of Cas9 degradation by dTAG-47. (A) Schematic showing the chemogenetic system to control Cas9 longevity using the small molecule, dTAG-47. Cas9 is fused with multiple (FKBP)12 F36V domains and investigated for dTAG-induced proteasomal degradation. (B) Dose-dependent and dTAG-47-induced Cas9 degradation in HEK293T cells transiently transfected with NL-FKBP-Cas9. (C) Upper panel: schematic of eGFP knockout assay to investigate Cas9 activity. Lower panel: eGFP disruption in U2OS.eGFP.PEST cells nucleofected with 10 pmol of ribonucleoprotein (RNP). The matched gRNA and the mismatched gRNA (1–3) were incubated with Cas9 or NL-FKBP-Cas9 to form the RNP. (D) dTAG-47 dose-dependent degradation of NL-FKBP-Cas9 in U2OS.eGFP.PEST cells measured by Cas9 activity in the eGFP disruption assay. (E) Top panel: schematic of knock-in of HiBiT ssODNs into GAPDH locus in HEK293T CRBN −/– and CRBN +/+ cell lines. GAPDH-HiBiT fusion protein will form a split NanoLuc protein upon complementation with LgBiT. Bottom panel: dTAG-47 dose-dependent (0, 1, 10, 100, 1000, 3000 nM) decrease in luminescence activity in CRBN +/+ cells but no change in the luminescence levels in the CRBN −/– cells, indicating that CRBN mediates Cas9 degradation. (F) Upper panel: schematic of eGFP knockout assay in Drosophila ’s S2 cells by measuring the indels using a T7E1 assay to investigate Cas9 activity. Lower panel: dose-dependent decrease in the indel formation due to degradation of NL-FKBP-Cas9 in S2 cells.

Article Snippet: For plasmid transfection, approximately 20 000 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid and 30 ng of gRNA expression plasmid using an SE Cell line 4D-Nucleofector X kit (Lonza) following the manufacturer’s protocol.

Techniques: Transfection, Knock-Out, Activity Assay, Incubation, Knock-In

Journal: ACS Central Science

Article Title: Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

doi: 10.1021/acscentsci.0c00129

Figure Lengend Snippet: Cas9 lifetimes impact DNA repair outcomes. (A) Deep-sequencing analysis of non-MH deletions and MH deletions, both raised from the NHEJ pathway. The mESC cell line with stable Reduced Library genomic integration was transfected with NL-FKBP-Cas9 plasmid. Then, 1 μM dTAG-47 was added at different time points after transfection (0–48 h) before genomic DNA was extracted at 120 h post-transfection. (B) ddPCR quantification of single-nucleotide exchange at the RBM20 locus in HEK293T cells following templated DNA repair. For this, 400 ng of NL-FKBP-Cas9 plasmid, 400 ng of RBM20 gRNA plasmid, and 40 pmol of ssODN were transfected to 0.2 × 10 6 HEK293T cells. dTAG-47 was introduced 0.5, 1, 2, 4, 8, 12, 24, and 48 h after transfection, and this was incubated until 72 h post-transfection. Cells were harvested at 72 h post-transfection, and percentages of HDR and NHEJ in the genomic DNA were analyzed by ddPCR analysis. At 72 h, no dTAG-47 addition occurred. (C) Luminescence-based quantification of HiBiT knock-in at the GAPDH locus in HEK293T cells following templated DNA repair. For this, 400 ng of NL-FKBP-Cas9 plasmid, 40 ng of GAPDH gRNA plasmid, and 40 pmol of ssODN were transfected to 0.2 × 10 6 HEK293T cells. dTAG-47 was introduced at 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after transfection and was incubated until 72 h post-transfection. Cells were lysed at 72 h post-transfection and complemented with LgBiT protein to measure the luminescence. At 72 h, no dTAG-47 addition occurred.

Article Snippet: For plasmid transfection, approximately 20 000 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid and 30 ng of gRNA expression plasmid using an SE Cell line 4D-Nucleofector X kit (Lonza) following the manufacturer’s protocol.

Techniques: Sequencing, Transfection, Plasmid Preparation, Incubation, Knock-In