To disrupt the endogenous CD19 locus in Nalm6 cells (provided by M. Milone, originally obtained from DSMZ) and to create a cell line only expressing CD19-sortase or ?d TCR-sortase, two guide RNAs targeting the human CD19 locus were obtained (IDT, sequences in Supplementary Table 2) and 5 × 10^6 Nalm6 cells were electroporated with a total of 50 pM ribonucleoprotein (consisting of Cas9 (IDT) and single-guide RNA (sgRNA)) in a total volume of 20 µl of Lonza P3 buffer (P3 primary cell 4D-Nucleofector X kit S) with a Lonza 4D-Nucleofector Core Unit (pulse protocol EO115) according to the manufacturer’s protocol. After CD19 disruption, Nalm6 cells were cultured at 0.2–1 × 106 cells per ml in standard medium for 14 days before sorting CD19 negative cells by FACS (BD Biosciences AriaII). Initial disruption efficiency was greater than 90%, which increased to more than 99% after sorting. CD19 negative Nalm6 cells were transduced with target proteins (CD3?, CD3d, CD3?, CD3e, ?d TCR-sortase or CD19 sortase) and positive cells were enriched by FACS.

Bulk and CD8 CART production

To generate naive or effector CARTs, naive and effector T cells were isolated from bulk primary human T cells and were electroporated with mRNA encoding CARs. Naive T cells were isolated either with the Naive Pan T Cell Isolation Kit (Miltenyi Biotec, catalogue no. 130-097- 095) or with a positive selection of CD62L and subsequent negative selection for CD45RA+ cells. For the latter approach, cells were stained with anti-CD62L-PE (BioLegend, DREG-56, catalogue no. 304840) and enriched with the anti-PE MultiSort kit (Miltenyi Biotec, catalogue no. 130-090-757) and LS column (Miltenyi Biotec, catalogue no. 130-042- 401), with the flowthrough reserved for the isolation of effector T cells described below. CD62L+ cells were flushed out and separated from MultiSort MicroBeads using the MultiSort Release Reagent and centrifugation. CD45RA+CD62L+ cells were subsequently isolated by negative isolation using CD45RO MicroBeads (Miltenyi Biotec, catalogue no. 130-046-001) and two columns (Miltenyi LS). To isolate effector T cells of the same donor as the naive T cells, flowthrough from the first CD62L selection was added to the column (Miltenyi LD) for negative selection of CD62L- cells. More than 95% population purity (determined by flow cytometry) was used in the presented studies. Following isolation, naive and effector cells were electroporated with 10 µg mRNA/1 × 10^7 T cells encoding the CARs using Lonza 4D-Nucleofector Core Unit (pulse code EH115) according to the manufacturer’s protocol.

To disrupt the endogenous TCR, T cells were cotransduced with lentiviral CAR constructs and pCAT003, a lentivirus transfer plasmid encoding sgRNA targeting TRAC and gift from J. Doudna (Addgene plasmid no. 171628)63. Immediately following debeading, up to 4 × 10^6 T cells were electroporated with 50 pM of Cas9 as described above with the modification of pulse code EO115. TCR- cells were negatively selected using CD3 MicroBeads (Miltenyi Biotec, catalogue no. 130-097-043) and LD column according to the manufacturer’s protocol. To genetically disrupt IKZF1, 1 × 10^6 T cells were electroporated immediately following debeading with 50 pM Cas9 and 100 nM guide RNA (IDT, Supplementary Table 2) as described above, with the modification of pulse code EH115.