GoKawiila, EGFP knock-in efficiency at the ACTB locus in Jurkat E6-1 cells using Cas9 or KE1 RNPs. b, Live (left) and EGFP+ (right) T cell numbers following EGFP-P2A insertion at the ACTB locus in primary T cells 3–5 days after electroporation with Cas9 or KE1 RNPs. c, Long-term stability of EGFP integration at the ACTB locus by KNIT editing, showing EGFP+ percentages (left) and representative flow cytometry plots (right) in sorted populations on days 10, 17, and 24 post-electroporation. d-f, On-target indel rates in uninserted alleles (d), and relative IFNB1 (e) and NOXA (f) expression after EGFP-P2A insertion at the ACTB locus using Cas9 or KE1 RNPs. RT-qPCR was performed 24 h post-electroporation and normalized to the Cas9 group. g-i, Representative flow cytometry (g), live and CAR+ primary T cell numbers (h), and representative confocal images (i) following anti-CD19 CAR insertion at the ACTB locus using Cas9 or KE1 RNPs. Flow cytometry with surface staining was performed with equal fractions of each sample at day 8 after electroporation. Related to Fig. 5f. Scale bars (i), 100 μm. j, CAR-T cell numbers (day 8) following CAR insertion at the ACTB locus using KE1 RNP, KE1 mRNA or KE2 mRNA. Related to Fig. 5h. k, Long-term stability of CAR integration at the ACTB locus by KNIT editing, showing CAR+ percentages (left) and representative flow cytometry plots (right) in sorted CAR-T cells on days 19 and 26 post-electroporation. CAR+ cells were sorted on day 12. l, Off-target insertion rates after CAR integration at the ACTB locus using Cas9 or KE. OT1-OT3: Cas9 off-target insertion sites shown in Fig. 2i. OT1 and OT3 were most prominent in Cas9-mediated CAR-T engineering. m, Circos plots showing chromosomal translocation rates following CAR insertion at the ACTB locus in T cells using Cas9 or KE. ND: Not detected. n, Relative T cell numbers 8 days after electroporation of Cas9 mRNA, KE1 mRNA and KE2 mRNA for CAR insertion at the TRAC locus, normalized to Cas9. o, Time-course of relative EGFP fluorescence intensity of CD19-EGFP-expressing HEK293T target cells co-cultured with indicated CAR-T cells and untreated T cells. Target cell only: CD19-EGFP HEK293T cells without T cells. p, Quantification of fluorescence intensity variance of CAR expression in LV-EF1α-CD19BBz CAR T cells, ACTB-CD19BBz CAR-T cells and TRAC-CD19BBz CAR-T cells. CV: coefficient of variation. For c, g and k: control: untreated T cells. For a-f, h and j-p, bars/values show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: unpaired two-tailed Student’s t-test (a, b, d, e, f, and h) or one-way ANOVA with Tukey’s multiple-comparisons test (j, n and p). All experiments were replicated independently at least three times.
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