Novel Thrombotic Function of a Human SNP in STXBP5 Revealed by CRISPR/Cas9 Gene Editing in Mice

Abstract

Objective To identify and characterize the effect of a single nucleotide polymorphism (SNP) in the STXBP5 locus that is associated with altered thrombosis in humans. Genome wide association studies (GWAS) have identified numerous SNPs associated with human thrombotic phenotypes, but determining the functional significance of an individual candidate SNP can be challenging, particularly when in vivo modeling is required. Recent GWAS led to the discovery of STXBP5 as a regulator of platelet secretion in humans. Further clinical studies have identified genetic variants of STXBP5 that are linked to altered plasma von Willebrand factor (VWF) levels and thrombosis in humans, but the functional significance of these variants in STXBP5 is not understood. Approach and Results We used CRISPR-Cas9 techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. Mice carrying the orthologous human mutation (encoding p. N437S in mouse STXBP5) have lower plasma VWF levels, decreased thrombosis, and decreased platelet secretion compared to wild-type mice. This thrombosis phenotype recapitulates the phenotype of humans carrying the minor allele of rs1039084. Decreased plasma VWF and platelet activation may partially explain the decreased thrombotic phenotype in mutant mice. Conclusions Using precise mammalian genome editing, we have identified a human non-synonymous SNP rs1039084 in the STXBP5 locus as a causal variant for a decreased thrombotic phenotype. CRISPR-Cas9 genetic editing facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.

Polymorphisms in sex steroid receptors: From gene sequence to behavior

“…The gold standard experiment is to replace the specific polymorphism in the genome, then test for phenotypic change (Wray, 2013). This goal can currently be accomplished in mice with Cre-loxP knockins (Glatt & Lee, 2016; Robins, 2008); the future of this field likely lies with the newer CRISPR-Cas9 technology (Hsu et al, 2014; Irion et al, 2014; Zhu et al, 2016) in a variety of non-traditional models.…”

“…Models of other polymorphisms can be developed as well. With the advent of CRISPR-Cas9 technology, which not only simplifies gene editing but extends the technique to a wider variety of model organisms (Hsu et al, 2014; Irion et al, 2014), we should expect to see increased availability of animals expressing human variants (see Zhu et al, 2016).…”

CRISPR links to long noncoding RNA function in mice: A practical approach

“…This unexpected finding should alert vascular biologists to variations in lncRNA sequences that may have hidden, clinically-relevant functions worthy of dissection in the mouse. The lncRNASNP2 database (http://bioinfo.life.hust.edu.cn) contains a trove of lncRNA sequence variants that, if found in conserved sequences of an orthologous mouse lncRNA, could be modeled in the mouse with three-component CRISPR editing as reported for a vascularassociated coding SNP [135]. Of note, polymorphisms in the promoter region of the NEAT1 lncRNA result in reduced expression and protein binding making these potentially viable substitutions to study in the mouse [136].…”

Modeling human disease in rodents by CRISPR/Cas9 genome editing

“…For example, a human GWAS identified a potential pathological SNP (rs1039084 A > G) in the STXBP5 gene, regulator of platelet secretion in humans. This mutation was then reproduced by CRISPR in the mouse with the nearly same thrombosis phenotype allowing to confirm the causality of this SNP in human (Zhu et al 2017 ). Likewise, whole-genome sequencing was used to perform a GWAS in a population-based biobank from Estonia.…”