Humanized mice bearing CRISPR/Cas9 Disruption of Signal Transducer and Activator of Transcription 1 (STAT1) to Model Primary Immunodeficiency

Abstract

Background: The search for a single, pathogenic genetic variant in a patient suspected to have a monogenic inborn error of immunity (IEI) often reveals a multitude of rare variants of unknown significance (VUS). Distinguishing which VUS is disease-causing versus the irrelevant, rare variants from the genetic background is slow and difficult. Advances in gene editing technology, particularly CRISPR/Cas9, promise to accelerate the timeline for the development of single-variant animal models, thus affording an experimental system for validating new genes and their variants. Objective: We sought to demonstrate a proof-of-concept of using CRISPR/Cas9 in human hematopoietic stem cells (hHSC) to develop of humanized mice bearing a hematopoietic deficiency in signal transducer and activator 1 (STAT1). Methods: Using CRISPR/Cas9, we introduced indels into the STAT1 gene of hHSCs and implanted them into immunodeficient mice. The reconstituted immune systems were assessed by flow cytometry. Results: Mice transplanted with cells edited to eliminate STAT1 developed human immune systems with diverse cell phenotypes. Lymphocytes from these reconstituted mice showed low expression of STAT1 protein and diminished phosphorylation of STAT1 in response to interferon stimulation. These data mirror the impaired, but not abolished, response to interferons seen in human partial STAT1 deficiency. CRISPR/Cas9 genome editing techniques can be used to rapidly and inexpensively create functional, humanized models of primary immune deficiencies.