TGC and NHEJ PuroeGFP I-SceI trGFP I-SceI PuroeGFP Bsd H2-K CD8 I-SceI CD4 I-SceI CMV GFP -, H2-K +, CD8 -, CD4 - GFP + CMV CD4 CD4 + H2-K CD8 CD4 CMV.

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TGC and NHEJ PuroeGFP I-SceI trGFP I-SceI PuroeGFP Bsd H2-K CD8 I-SceI CD4 I-SceI CMV GFP -, H2-K +, CD8 -, CD4 - GFP + CMV CD4 CD4 + H2-K CD8 CD4 CMV CD8 + CD8 CD4 CM H2-K - 2-K H2-K - CD4/8 -

TGC and NHEJ PuroeGFP I-SceI trGFP I-SceI PuroeGFP Bsd H2-K CD8 I-SceI CD4 I-SceI CMV GFP -, H2-K +, CD8 -, CD4 - GFP + CMV CD4 CD4 + H2-K CD8 CD4 CMV CD8 + CD8 CD4 CM H2-K - 2-K H2-K - CD4/8 -

2.4% 14.5% 0.23%0.11% 0.37% 97.3% 84.4% 0.42% 0.29%0.003% 0.004% H2-K d (PE) GFPGFP Cell lineExperimentTargeting (%)H2-K d loss (%) among uncorrectedcorrected # #1# #2# # no I-SceI H2-K d stained + I-SceI stained H2-K d + I-SceI unstained A  B

9.7% 10.9% 0.77%0.67% 1.62% 89.4% 86.6% 1.96% 1.34%0% 0.001% H2-K d (PE) GFPGFP Cell lineExperimentTargeting (%)H2-K d loss (%) among uncorrectedcorrected # #1# #2# # no I-SceI H2-K d stained + I-SceI stained H2-K d + I-SceI unstained A  B