GoKawiilCell culture and nucleofection
K562 (CCL-243) and Jurkat (TIB-152) cells were obtained from American Type Culture Collection (ATCC) and cultured at 37 °C under 5% CO 2 in RPMI media (ThermoFisher Scientific, 11875093) supplemented with 10% FBS, and 1% Penicillin/Streptomycin (ThermoFisher Scientific, 15140122). HEK293T (CRL-1573) cells were obtained from the ATCC and cultured at 37 °C under 5% CO 2 in DMEM media (ThermoFisher Scientific, 11995065) supplemented with 10% FBS, and 1% penicillin/streptomycin. Cell lines were authenticated by the supplier and tested negative for mycoplasma.
For standard K562 nucleofections, 2 × 105 cells were electroporated with 750 ng pCMV-PE7 (ref. 45) (Addgene #214812), 250 ng of each standard pegRNA16 vector (derived from Addgene #132777), and the indicated concentration of PA donor with an Amaxa 4D-nucleofector (Lonza) using the SF cell line nucleofection kit (Lonza, V4XC-2032) (pulse FF-120). For each nucleofection, cells were resuspended in 20 µl of nucleofection buffer, the indicated amount of DNA was added while maintaining a final total volume of less than 24 µl, and nucleofection mixes were transferred to the 16-well strip. For short synthetic ssDNA donors, the optimal concentrations were 800 nM to 1,600 nM of each ssDNA donor. These concentrations could not be achieved with long ssDNA donors due to toxicity, and 100 nM to 200 nM were used. For standard 3′-odsDNA and dsDNA donors, 78 nM to 159 nM were used. Finally, for larger 3′-odsDNA and dsDNA donors ranging from 3.1 to 12.1 kb, 13 nM to 53 nM were used. Where indicated, the F + E scaffold modifications61 were included in the pegRNAs. For initial experiments (Fig. 1 and Extended Data Figs. 1 and 2), K562 cells were electroporated with 750 ng pCMV-PEmax44 (Addgene #174820), 250 ng of each tevopreq1-epegRNA62 vector (derived from Addgene #174038) harbouring the (F + E) scaffold modifications61, and the indicated concentration of PA donor. For nuclease PA experiments, the HNH domain of PE7 (Addgene #214812) was restored to generate pCMV-PE7nuclease via Gibson assembly. For 53BP1 inhibitor overexpression under the EF1α promoter, the hMLH1dn cassette of pEF1a-hMLH1dn (Addgene #174824) was replaced with the i53 (ref. 63) coding sequence. For HDR experiments, K562 cells were electroporated with 750 ng pX330-U6-Chimeric_BB-CBh-hSpCas9 (ref. 64) (Addgene #42230) expressing the sgRNA of interest and 16 pmol ssDNA donor. All high-quality plasmids used for electroporation were purified using the EZNA FastFilter Plasmid DNA Midi Kit (Omega Bio-tek, D6905-04) and DNA concentration and purity was assessed by nanodrop. The composition of all nucleofection mixes is provided in the Supplementary Information.
For Jurkat nucleofections, 1 × 106 cells were electroporated with 500 ng pCMV-PE7, 250 ng of each standard pegRNA, and the indicated concentration of PA donor using the SE cell line nucleofection kit (Lonza, V4XC-1302) (Pulse CL-120). For HEK293T nucleofections, 2 × 105 cells were electroporated with 750 ng pCMV-PE7, 375 ng of each standard pegRNA, and the indicated concentration of PA donor using the SF cell line nucleofection kit (pulse CM-130). For benchmarking experiments, 750 ng of pCMV-PE7, 750 ng eeBxb1 (ref. 23) (Addgene #222339), or 750 ng pX330 vector was used for K562 and HEK293T cells, and 500 ng of each editor expression vector was used for Jurkat cells. The total concentration of DNA was normalized between PA and PASSIGE while maintaining the original vector ratio for the latter23. The composition of all nucleofection mixes is provided in the Supplementary Information.
StemSelect PD-0332991 (Sigma, 5304870001) was dissolved at 10 mM in water and stored at −80 °C. Where indicated, K562 cells were treated with 5 µM PD-0332991. Ouabain octahydrate (Sigma, O3125-250GM) was dissolved at 5 mg ml−1 in water, and working dilutions were prepared in water and stored at −20 °C. Where indicated, ouabain selection was performed with 0.5 µM 3 days post-nucleofection until all non-resistant cells were eliminated. Puromycin (Sigma, P8833-25MG) was dissolved at 1 mg ml−1 in water and stored at −20 °C. Where indicated, puromycin selection was performed with 1 µg ml−1 3 days post-nucleofection until all non-resistant cells were eliminated. AZD7648 (MedChemExpress, HY-111783) and PolQi1 (MedChemExpress, HY-159078) were dissolved at 10 mM in DMSO, and working dilutions were prepared in water and stored at −80 °C. Where indicated, K562 and HEK293T cells were treated during 3 days post-nucleofection with 1 µM AZD7648 and 1.5 µM PolQi1. Jurkat cells were treated during 3 days post-nucleofection with 0.5 µM AZD7648 and 0.5 µM PolQi1.
Primary CD34+ HSPC culture and nucleofection
Cryopreserved human CD34+ HSPCs from mobilized peripheral blood of deidentified healthy donors were obtained from the Fred Hutchinson Cancer Research Center (Seattle, Washington) and their use was determined as exempt from human subjects research requirements by Boston Children’s Hospital Institutional Review Board. CD34+ HSPCs were cultured in X-Vivo-15 media (Lonza, 04-418Q) supplemented with 100 ng ml−1 human Stem Cell Growth Factor (SCF) (R&D Systems, 255-SC-010), 100 ng ml−1 human thrombopoietin (TPO) (Peprotech, 300-18), and 100 ng ml−1 recombinant human FMS-like Tyrosine Kinase 3 Ligand (Flt3-L) (Peprotech, 300-19). CD34+ HSPCs were thawed and cultured for 24 h in the presence of cytokines, and electroporated using the P3 Primary Cell X kit S (Lonza, V4XP-3032) according to the manufacturer’s recommendations. Cells (2.5 × 105) were electroporated with 2,000 ng PE7 mRNA45, an equimolar ratio of simian immunodeficiency virus (SIV) Vpx mRNA50, and the indicated concentration of each pegRNA and ssDNA donors using pulse code DS-130. Following electroporation, 80 µl of media supplemented with cytokines was added to each well and cells were incubated for 10 min prior to transfer to the culture plate. Cells were cultured in a 48-well plate in a final volume of 500 µl of media supplemented with 50 µM of each deoxynucleoside50. Cell viability was assessed 24 h post-nucleofection via Trypan Blue staining and manual counting using a haemocytometer, and genomic DNA was purified 3 days post-nucleofection. Deoxynucleosides (dA, Sigma-Aldrich, D8668; dG, Sigma-Aldrich, D0901; dC, Sigma-Aldrich, D0776; and dT, Sigma-Aldrich, T1895) were resuspended in water at 12.5 mM each, filter-sterilized, and stored at −20 °C.
Primary CD3+ T cell culture and nucleofection
Human CD3+ T cells were isolated from leukocyte reduction system (LRS) cones of deidentified healthy donors from the Blood Donor Center at Boston Children’s Hospital and their use was determined as exempt from human subjects research requirements by Boston Children’s Hospital Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) were collected via density gradient centrifugation by layering the blood diluted with PBS on Ficoll-Paque (Cytiva, 17144002) using SepMate-50 tubes (StemCell Technologies, 85450). PBMCs were aspirated and washed with cold PBS. Bulk T cells were isolated by magnetic labelling with CD3 MicroBeads (Miltenyi Biotec, 130-050-101) and separation via LS columns (Miltenyi Biotec, 130-042-401) using a manual MACS separator according to the manufacturer’s recommendations. CD3+ T cells were either used fresh or cryopreserved.
Primary CD3+ T cells were cultured at a density of 106 cells per ml in ImmunoCult T Cell Expansion Medium (StemCell Technologies, 10981), supplemented with 1% penicillin/streptomycin at 37 °C with 5% CO 2 . CD3+ T cells were activated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator (25 µl per million cells) (StemCell Technologies, 10990) for 24 h and cultured with 300 U ml−1 IL-2 (StemCell Technologies, 78036.1). Resting T cells were kept in culture with 1 ng ml−1 IL-7 (Miltenyi Biotec, 130-095-367) and 1 ng ml−1 IL-15 (Miltenyi Biotec, 130-095-760). Primary T cells were electroporated using the P3 Primary Cell X kit S (Lonza, V4XP-3032) according to the manufacturer’s recommendations. Cells (1.5 × 106) were electroporated with 2,000 ng PE7 mRNA45, an equimolar ratio of SIV Vpx mRNA50, 200 pmol of each pegRNA, and 4 pmol of each ssDNA donor using pulse code DS-137. Following electroporation, 80 µl of media supplemented with cytokines was added to each well and cells were incubated for 10 min prior to transfer to the culture plate. Cells were cultured in a 48-well plate in a final volume of 500 µl of media supplemented with 50 µM of each deoxynucleoside50.
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