Construction of FACS reporter cell line and FACS-based genome-scale CRISPRi screen

Seven days after transduction, 3.2?×?10^8 fully selected cells (K562 CRISPRi cells) were nucleofected using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF-120, according to the manufacturer’s protocol. Each nucleofection consisted of 1?×?10^7 cells, 7,500?ng pCMV-SaPE2 (Addgene, 174817)5 , 2,500?ng +7 GG-to-CA pegRNA plasmid and 833?ng +50 nicking sgRNA plasmid. Three days after nucleofection, 1.5?×?10^8 cells were sorted using a BD FACSAria Fusion flow cytometer. 

Tissue culture transfection and transduction protocols and gDNA extraction

For prime editing in Lenti-X 293T, HeLa and U2OS cells by plasmid nucleofection, 750?ng prime editor plasmid, 250?ng pegRNA plasmid and 83?ng nicking sgRNA plasmid (PE3 and PE5) were nucleofected. For each sample, 2?×?10^5 LentiX-293T cells, 1?×?10^5 HeLa cells or 1?×?10^5 U2OS cells were nucleofected using SF (Lonza, V4XC-2032), SE (Lonza, V4XC-1032) and SE Cell Line 4D-Nucleofector X kit S with program CM-130, CN-114 and DN-100, respectively, according to the manufacturer’s protocols. PE4 and PE5 experiments in U2OS cells were performed with pCMV-PEmax-P2A-hMLH1dn and pCMV-PE7-P2A-hMLH1dn editor plasmids. After nucleofection, cells were cultured in 24-well plates (Greiner Bio-One, 662165), and the culture medium was removed 72?h after nucleofection. 

For prime editing in K562 and U2OS cells using editor mRNA and  synthetic pegRNA, 1?×?10^6 K562 and 1?×?10^5 U2OS cells were nucleofected  with 1?µg editor mRNA and 50?pmole synthetic pegRNA using the SE Cell Line 4D-Nucleofector X kit S (Lonza, V4XC-1032) with program FF-120 and DN-100, respectively, according to the manufacturer’s protocols. After nucleofection, cells were cultured for 72?h and collected  for gDNA extract. 

Generation of K562 clones with PEmax knock-in at AAVS1
A total of 91.5?pmole Alt-R S.p. Cas9 Nuclease V3 (Integrated DNA Technologies, 1081058) and 150?pmole custom Alt-R gRNA targeting  AAVS120 (Integrated DNA Technologies) (Supplementary Table 8) were complexed for 20?min at room temperature and were nucleofected  together with 2,000?ng AAVS1 PEmax knock-in plasmid as the HDR template into 7.5?×?10^5  K562 cells using the SE Cell Line 4D-Nucleofector X  kit (Lonza, V4XC-1032) and program FF-120, according to the manufacturer’s protocol. Four days after nucleofection, cells were selected using 400?µg?ml-1 geneticin (Gibco, 10131027) for 2?weeks before sorted using  a BD FACSAria Fusion flow cytometer into 96-well plates at 1?cell per well with 150?µl conditioned culture medium. Single cells were grown  and expanded for 2–3?weeks into clonal lines, from which the one with  the highest and most homogenous eGFP expression by AttueNXT flow cytometry analysis was selected as the K562 PEmax parental cell line.

T cell isolation, culture and prime editing

Human peripheral blood Leukopaks enriched for peripheral blood  mononuclear cells were sourced from StemCell (StemCell Technologies, 200-0092) with approved StemCell institutional review board  (IRB). No preference was given with regard to sex, ethnicity or race. Use  of de-identified cells is considered exempt human subjects research  and is approved by the UCSF IRB. T?cells were isolated using the EasySep  Human T?cell isolation kit (StemCell Technologies, 100-0695) according to manufacturer’s instructions. Immediately after isolation, T?cells  were used directly for in vitro experiments. All T?cells were cultured in  complete X-VIVO 15 consisting of X-VIVO 15 (Lonza Bioscience, 04-418Q)  supplemented with 5% FBS (R&D systems), 4?mM N-acetyl-cysteine (RPI,  A10040) and 55?µM 2-mercaptoethanol (Gibco, 21985023). Pan CD3+ T?cells were activated with anti-CD3/anti-CD28 Dynabeads (Gibco,  40203D) at a 1:1 bead-to-cell ratio in the presence of 500?IU?ml-1 IL-2.  Two days after stimulation, T?cells were magnetically de-beaded and  taken up in P3 buffer with supplement (Lonza Bioscience, V4SP-3096) at 37.5?×?10^6  cells per ml. Next, 1.5?µg PEmax or PE7 mRNA mixed with  50?pmole synthetic pegRNA (Integrated DNA Technologies; Supplementary Table 8) was added per 20?µl cells, not exceeding 25?µl total 
volume per reaction. Cells were subsequently electroporated using  a Lonza 4D Nucleofector with program DS-137. Immediately after  electroporation, 80?µl warm complete X-VIVO15 was added to each  electroporation well, and cells were incubated for 30?min in a 5% CO2 incubator at 37?°C followed by distribution of each electroporation  reaction into 3 wells of a 96-well round-bottom plate. Each well was  brought to 200?µl complete X-VIVO 15 and 200?IU ml–1 IL-2. Cells were  subcultured and expanded through the addition of fresh medium and  IL-2 every 2–3?days. Four days after electroporation, approximately  5?×?10^5  cells were spun down at 500g for 5?min, and gDNA was extracted using a DNeasy Blood & Tissue kit (Qiagen, 69506) per the manufacturer’s instructions with an elution volume of 100 µl. 

HSPC isolation, culture and prime editing

CD34+HSPCs were cultured with  X-Vivo-15 medium supplemented with 100?ng?ml-1 human stem cell  growth factor, 100?ng?ml-1 human thrombopoietin and 100?ng?ml-1
recombinant human FMS-like tyrosine kinase 3 ligand. CD34+  HSPCs  were thawed and cultured for 24?h in the presence of cytokines before  nucleofection. Overall, 2.5?× 10^5 CD34+ HSPCs were electroporated  using a P3 Primary Cell X kit S (Lonza Bioscience, V4SP-3096) according to the manufacturer’s recommendations with 2,000?ng PEmax or  PE7 mRNA and 200?pmole synthetic pegRNA or epegRNA using pulse code DS-130.