Figure 1.

Multiplexed CRISPR editing of CLL-LOF lesions generates CLL disease models. A, Schema for the generation of transplant lines. BM donor lines containing homozygous del(13q) and B cell–restricted Cas9-GFP [i.e., del(13q)-Cd19Cas9-GFP] were obtained by intercrossing del(13q)-Cd19cre/cre and del(13q)-Cas9/Cas9 mice. LSKs isolated from 8- to 12-week-old del(13q)-Cd19Cas9 animals were transduced in vitro with a pool of lentivirus-expressing sgRNAs against the 6 or 5 LOF mutations of interest (Atm, Mga, Samhd1, Chd2, Birc3, Trp53 in “6-plex”; Trp53 absent in “5-plex”) or corresponding nontargeting pools of scrambled sgRNAs and transplanted into 8- to 12-week-old immune-competent (CD45.1) or immune-deficient (NSG) recipients (n = 30–35/group). B, Single-cell qPCR-based analysis of sgRNA expression in LSK from the 3 targeting and the 3 nontargeting cohorts, as assessed at 72 hours after transduction and in vitro culture. The number of sgRNAs quantified in individual single cells is displayed. C, Percent (%) animals with circulating CLL in targeting and nontargeting cohorts. P, Fisher exact test. D, Longitudinal tumor burden assessments by flow-cytometric analysis of peripheral bleeds. A logistic mixed-effects model was used to estimate the average trajectories of peripheral longitudinal tumor burden (%GFP⁺mCh⁺B220⁺CD5⁺Igκ⁺ cells) in individual mice from the targeting (6-plex: dark brown; 5-plex: salmon) and nontargeting cohorts (gray). Disease onset and survival (median and range) for each cohort are indicated.