Apr 22, 2024

Public workspaceMitochondrial DNA base editing in HEK293T cells

DOI
dx.doi.org/10.17504/protocols.io.yxmvm3rnol3p/v1
  • 1Department of Genetics, Yale School of Medicine, New Haven, CT, USA.
Justin Cohen
Open access
DOI: dx.doi.org/10.17504/protocols.io.yxmvm3rnol3p/v1
Protocol Citation: Nicole Lake, Kaiyue Ma, Justin Cohen, Monkol Lek 2024. Mitochondrial DNA base editing in HEK293T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3rnol3p/v1
Manuscript citation:
Lake NJ, et al. Quantifying constraint in the human mitochondrial genome. bioRxiv (2023). https://doi.org/10.1101/2022.12.16.520778
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 12, 2024
Last Modified: April 22, 2024
Protocol Integer ID: 95136
Keywords: DdCBE, Base Editor, Mitochondria, Transfection, HEK293
Abstract
This protocol is for the transfection of mitochondrial-targeted DddA-derived cytosine base editors (DdCBE), and their subsequent selection, in HEK293T cells. This uses a dual plasmid system, where a ‘left’ and ‘right’ DdCBE are needed for editing. Enrichment of cells with both DdCBE halves is achieved by separate drug selection for the left and right plasmids.
Materials
HEK293 cells (ATCC, CRL-3216)
HEK media
  • DMEM DMEM (Gibco, 11965092)
  • 10% FBS (R&D Systems S11150)
  • No antibiotics
DdCBE plasmids:
• Left/left-dead (blastR) and Right (PuroR)
Lipofectamine 3000 reagents (ThermoFisher, L3000008)
Opti-Mem I reduced serum medium (ThermoFisher, 31985070)
12-well tissue culture plates (Corning, 353043)
Blasticidin (ThermoFisher, A1113903)
Puromycin (ThermoFisher, A1113803)
Protocol materials
ReagentHEK293TATCCCatalog #CRL-3216
Step 1
ReagentBlasticidin S HCl (10 mg/mL)Thermo FisherCatalog #A1113903
In 2 steps
ReagentDMEM, high glucoseThermo Fisher ScientificCatalog #11965092
In 4 steps
ReagentFetal Bovine Serum (FBS)ATCCCatalog #30-2020
In 4 steps
ReagentOpti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985070
In 2 steps
ReagentLeft DdCBE PlasmidaddgeneCatalog #179682
Step 2.2
ReagentPuromycin DihydrochlorideThermo FisherCatalog #A1113803
In 2 steps
ReagentLipofectamine™ 3000 Transfection ReagentThermo Fisher ScientificCatalog #L3000008
In 3 steps
ReagentFalcon® 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Individually WrCorningCatalog #353043
Step 1.2
ReagentRight DdCBE PlasmidaddgeneCatalog #179686
Step 2.2
ReagentLeft Dead (inactive) DdCBE PlasmidaddgeneCatalog #179683
Step 2.2
Plating of the HEK293T Cells
Plating of the HEK293T Cells
Plating of ReagentHEK293TATCCCatalog #CRL-3216

The day before transfection, trypsinize and count the cells.
In a ReagentFalcon® 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Individually WrCorningCatalog #353043 , plate 150000 cells per well in Amount1 mL per well of complete HEK media (ReagentDMEM, high glucoseThermo Fisher ScientificCatalog #11965092 with Concentration10 % volume ReagentFetal Bovine Serum (FBS)ATCCCatalog #30-2020 without antibiotics).
Note
Antibiotics can reduce transfection efficiency and were thus omitted.

Wait for cells to attach DurationOvernight at Temperature37 °C in a 5% CO2 tissue culture incubator.

Transfection of DdCBE Plasmids
Transfection of DdCBE Plasmids
Transfection of DdCBE Plasmids
For each well of cells to be transfected, dilute Amount3 µL of ReagentLipofectamine™ 3000 Transfection ReagentThermo Fisher ScientificCatalog #L3000008 into Amount50 µL (total volume) of ReagentOpti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985070 and mix well.

In a separate tube, for each well of cells to be transfected, dilute Amount2 µg of each DdCBE plasmid ReagentLeft DdCBE PlasmidaddgeneCatalog #179682 orReagentLeft Dead (inactive) DdCBE PlasmidaddgeneCatalog #179683 with ReagentRight DdCBE PlasmidaddgeneCatalog #179686 that had been modified to include PuroR marker (left and right, for total of Amount4 µg plasmid DNA (pDNA))
Note
The DdCBE plasmids used were obtained from Addgene, which included left (Addgene #179682) and right (Addgene #179686) DdCBE plasmids for editing, and a left dead (i.e. inactive) DdCBE plasmid (Addgene #179683) used with the right as a control. For this protocol, the right DdCBE plasmid was modified by replacing the BSD gene with PuroR to enable dual selection. Please see the associated publication for more plasmid details.
into Amount50 µL (total volume) of ReagentOpti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985070 . Then add Amount8 µL P3000 Reagent from ReagentLipofectamine™ 3000 Transfection ReagentThermo Fisher ScientificCatalog #L3000008 (a 2:1 ratio to DNA) directly to the diluted pDNA. Mix well.

Add the diluted pDNA solution in P3000 reagent (from 2.2) to diluted ReagentLipofectamine™ 3000 Transfection ReagentThermo Fisher ScientificCatalog #L3000008 (from 2.1), mix, and incubate forDuration00:15:00 min at room temperature.

15m
Discard the old medium in the well. Add Amount1 mL complete HEK media (ReagentDMEM, high glucoseThermo Fisher ScientificCatalog #11965092 with Concentration10 % volume ReagentFetal Bovine Serum (FBS)ATCCCatalog #30-2020 without antibiotics) to each tube, mix well, and add to the corresponding well. Do this step well by well. Incubate the cells at Temperature37 °C in a 5% CO2 tissue culture incubator.

Selection of the Transfected Cells
Selection of the Transfected Cells
18h
Selection of the Transfected Cells
Duration18:00:00 hrs later, replace the medium with complete HEK media (ReagentDMEM, high glucoseThermo Fisher ScientificCatalog #11965092 with Concentration10 % volume ReagentFetal Bovine Serum (FBS)ATCCCatalog #30-2020 without antibiotics) containing up to Amount5 ug/mL of ReagentBlasticidin S HCl (10 mg/mL)Thermo FisherCatalog #A1113903 and Amount1 ug/mL of ReagentPuromycin DihydrochlorideThermo FisherCatalog #A1113803
18h
Continue selection for 10-14 days and replace the medium every 2 days with complete HEK media containing ReagentBlasticidin S HCl (10 mg/mL)Thermo FisherCatalog #A1113903 and ReagentPuromycin DihydrochlorideThermo FisherCatalog #A1113803 . Ensure drug selection is not finished until all the control untransfected cells are dead. Passage the cells to a larger well-size or flask if needed.
Once selection is finished, maintain the cells in complete HEK media (ReagentDMEM, high glucoseThermo Fisher ScientificCatalog #11965092 with Concentration10 % volume ReagentFetal Bovine Serum (FBS)ATCCCatalog #30-2020 without antibiotics) for at least 2 days before performing any experiments.
Protocol references
Protocol adapted from Mok, B.Y., et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020), and Mok, B.Y., et al. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA. Nat Biotechnol 40, 1378–1387 (2022).