Improved cytosine base editors generated from TadA variants

Lam DK, Feliciano PR, Arif A, Bohnuud T, Fernandez TP, Gehrke JM, Grayson P, Lee KD, Ortega MA, Sawyer C, Schwaegerle ND, Peraro L, Young L, Lee SJ, Ciaramella G, Gaudelli NM.
Source: Nat Cell Biol
Publication Date: (2023)
Issue: :
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems

Isolation and culture of allogeneic human T cells

Human T cells were isolated from leukapheresis products (Leukopaks, HemaCare) by positive selection using CD4 and CD8 MicroBeads (Miltenyi,130045101 and 130045201). T cells were frozen at 25–50 × 10^6 cells per ml of Cryostor CS10 (Stemcell Technologies, 1001061).

For editing experiments, T cells were thawed in a water bath at 37 °C and then allowed to rest overnight in ImmunoCult-XF T Cell Expansion Medium containing (Stemcell Technologies, 10981) 5% CTS Immune Cell SR, Glutamax, 10 mM HEPES, 1% Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122). The next day, T cells were activated using 25 µl of ImmunoCult Human CD3/CD28/CD2 T Cell Activator (Stemcell Technologies, 10970) per ml of cells at 1 × 10^6 cells per ml plus 300 IU/ml of IL-2 (CellGenix, 1420050). Fresh IL-2 was added to T cells every 2–3 d. T cells were cultured at 37 °C and 5% CO2.

Electroporation of human T cells

T cells were transfected 72 h after activation. Cells were resuspended in P3 Primary Cell Nucleofector Solution containing Supplement 1 (Lonza, V4SP-3960). 1 × 10^6 T cells were edited with 1 µg of synthetic sgRNA (IDT) and 2 µg of editor mRNA in a total volume of 20 µl using P3 96-well Nucleocuvette kit (Lonza, V4SP-3960). The three sgRNAs used are as follows: B2M Exon 2 (B2M Ex.2), pmSTOP C6, CD247 pomSTOP C7 and PD-1 Ex.1 SA C7 are specified in Supplementary Table 3. T cells were electroporated with the 4D-Nucleofector system (Lonza, AAF-1003B and AAF-1003S) using program DH-102. All experiments were performed with two independent T cell donors. For NGS analysis, 1×10^5 T cells per condition at each timepoint were pelleted, supernatant was removed and pellets were resuspended in 50 ml of QuickExtract DNA Extraction buffer (Lucigen, QE09050) and transferred to a PCR plate for targeted amplicon sequencing. 


Cytosine base editors (CBEs) enable programmable genomic C·G-to-T·A transition mutations and typically comprise a modifed CRISPR–Cas enzyme, a naturally occurring cytidine deaminase, and an inhibitor of uracil repair. Previous studies have shown that CBEs utilizing naturally occurring cytidine deaminases may cause unguided, genome-wide cytosine deamination. While improved CBEs that decrease stochastic genome-wide of-targets have subsequently been reported, these editors can sufer from suboptimal on-target performance. Here, we report the generation and characterization of CBEs that use engineered variants of TadA (CBE-T) that enable high on-target C·G to T·A across a sequence-diverse set of genomic loci, demonstrate robust activity in primary cells and cause no detectable elevation in genome-wide mutation. Additionally, we report cytosine and adenine base editors (CABEs) catalyzing both A-to-I and C-to-U editing (CABE-Ts).Together with ABEs, CBE-Ts and CABE-Ts enable the programmable installation of all transition mutations using laboratory-evolved TadA variants with improved properties relative to previously reported CBEs.