Puzzling Low Surface Expression of H-DreI-B7 in IgL-targeted Clones Dre #4Dre #3Dre #2 Dre #3 Dre #2 IgM-FITC HA-APC dsOligo-Biotin/SAv-PE Cell count (%)

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Puzzling Low Surface Expression of H-DreI-B7 in IgL-targeted Clones Dre #4Dre #3Dre #2 Dre #3 Dre #2 IgM-FITC HA-APC dsOligo-Biotin/SAv-PE Cell count (%) unstaineddsDre4dsAni1 Dre #4 DT40 Ψ-/- AID R DT40 TetR WT

Hypermutation optimized-DreI-B7/Hypermut2 clones 3D5 3F4 HDreI5A8 HDreI3F5 HDreI5F8 HDreI 5B8 HDreI 5B12 HDreI 3 weeks in culture: 1 week in culture: I-AniI/Hypermut2 control Better surface expression, but no integration into rearranged IgL allele IgM-FITC HA-tag-APC

DT40 H-DreI C4 HyperDreI 5A8HyperDreI 5F8HyperDreI 4E12HyperDreI 5E6 dsDre4-BT:SAv-PE Cell count (% of maximum) Good recognition of H-DreI target sequence Control cells dsDre4 staining corresponds well to HA-tag staining unstaineddsDre4

Flow cytometry analysis of CTLMA2 target 5’ TGAGGAGGTTTCTCTGTAA 3’ 5’ AGAGGACCTTCATCTGTCA 3’ I-AniI WT: I-AniI CTLMA2: MFl B10 MFl DT40 MFl DT40 = 1.0

DT40 B3 I-AniI B10 I-AniI 3D5 I-AniI dsAni1 wtdsAni1 +1C+2A dsOligo-Alexa647 Cell count (% of maximum) Flow cytometry cleavage assay for CTLMA2 target