Individual CLL LOF lesions alter mature B-cell survival but are insufficient to drive leukemia in mouse models. (A) Transplant schema and generation of singly-edited mouse lines. LSKs were lentivirally transfected in vitro with sgRNAs targeting the single LOFs (or a non-targeting scramble control, ie, Nt-1) in addition to the mCherry marker before the engraftment into sublethally irradiated CD45.1 recipient mice. PCR-based targeted deep sequencing of DNA from peripheral blood edited B cells (ie, GFP⁺mCherry⁺) was used to confirm presence of ∼45% to 85% gene edits (>70% frameshift mutations) across the 6 genes. (B) In vitro survival of normal B cells isolated from the spleen of 5 animals per group (6 LOF-expressing strains and 1 Nt-1) and cultured in vitro in presence of LPS+IL4 for 3 days. Fold increase over day 0 is displayed alongside representative flow cytometric plots from 1 Nt-1 and 1 Trp53-depleted sample. P-value lower or equal to 0.05, ANOVA with Dunnett’s correction for multiple comparisons. (C) Percent (%) GFP⁺mCherry⁺ B cells as assessed on longitudinal bleeds over the course of 24 months, in 10 animals per group. ∗P-value lower or equal to 0.05; lower or equal to 0.05; RM-ANOVA, repeated measures analysis of variance.