Using Multiplexed Genome Editing to Distinguish between Loss- and Gain-of-Function Variants in CARD11

Genetic variants in CARD11 contribute to several diseases caused by dysregulation of the adaptive immune system. Dominant-negative, loss-of-function and gain-of-function variants in CARD11 variants lead to primary immunodeficiency disease. However, primary immunodeficiency disease caused by CARD11 gain-of-function variants often progresses to B-cell malignancy. The clinical course and treatment options depend on the type of CARD11 mutation. Unfortunately lymphocyte immunophenotyping and traditional proliferation assays do not distinguish the variant effect or predict the likelihood of malignancy. To address this clinical challenge, we coupled multiplexed genome editing to a cell growth assay that is read out by DNA sequencing in order to assess functional effects of thousands of CARD11 variants in parallel. By co-delivering CRISPR/Cas9 ribonucleoprotein complexes with libraries of single-stranded oligonucleotide repair templates, we generated lymphoma cell populations containing all possible single-nucleotide variants (~2400 different protein coding changes) in the N-terminal 140 amino acids of CARD11. Due a requirement for CARD11 in these lymphoma cells, those with dominant-negative and loss-of-function CARD11 variants grow more slowly in the absence of treatment, whereas those with gain-of-function variants grow faster in the presence B cell receptor pathway inhibitors. By tracking the relative abundance of each variant in the population by next generation sequencing over multiple conditions, we determined the functional effect of each. Finally, we evaluated previously un-described dominant-negative, loss-of-function and gain-of-function variants during differentiation of primary human B cells and NF-kB signaling. The results of our experiments demonstrate the utility of multiplexed functional assays for determining variant effect in proteins where distinguishing between dominant-negative, loss- and gain-of-function effects are required to guide diagnoses and treatment.

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