Inactivation of Each of the Duplicated Hba Genes in Mice using a Stochastic CRISPR‐Cas9 Approach

Alpha globin regulates nitric oxide signaling in resistance arteries by binding to endothelial nitric oxide synthase and catalyzing a reaction between nitric oxide and oxygen. In both mice and humans, alpha globin is expressed by duplicated gene loci on chromosome 16. In mice, the amino acid sequences of both genes are identical, and the transcripts of Hba1 and Hba2 differ by only a single base pair difference in exon 3. Using locus‐specific reverse transcriptase droplet digital PCR, we recently discovered that in mice, the Hba1 and Hba2 genes are differentially expressed in vascular tissue compared to blood. In blood, Hba1 transcripts are 10 times more abundant than Hba2 transcripts; conversely, in isolated perfused resistance arteries, Hba2 is 1.6 times more abundant than Hba1 . Thus, Hba1 and Hba2 genes appear to be under different transcriptional control in endothelial cells compared to erythrocyte progenitors. We sought to exploit the relative differences in Hba1 and Hba2 gene expression to generate global knock‐out mice that would have decreased expression of alpha globin in the vascular endothelium while maintaining adequate expression in the erythrocyte progenitors – potentially generating a vascular phenotype without the confounding effects of anemia. Initial attempts to selectively target Hba2 using guide RNAs that recognized Hba2 ‐specific flanking sequences failed. As an alternative approach, we used guide RNAs that recognized sequences common to the transcriptional start sites of both Hba1 and Hba2 , and then titrated the activity of the Cas9 enzyme to generate targeted deletions of one or the other gene randomly. This stochastic approach produced 4 pups with a deletion in Hba1 , 3 pups with a deletion in Hba2 , and 5 pups with deletions in both Hba1 and Hba2 , out of a total of 40 live‐born pups, as detected by size polymorphism of locus‐specific endpoint PCR. Each PCR band was cut out of the gel and the DNA purified for Sanger sequencing. Sequencing revealed the deletions ranged in size from 175 to 505 bp and included the transcriptional start of the gene in all instances. A total of 9 mice (3 Hba1 , 3 Hba2 , and 3 with deletions in both genes) were backcrossed with the C57BL/6 background strain to obtain heterozygous F1 mice. F1 mice with identical genotypes will be interbred to generate F2 homozygous mice. Initial phenotyping will include measurement of Hba1 and Hba2 locus‐specific gene expression in both whole blood and vascular tissue, measurement of red cell indices to assess for microcytic anemia, and ex vivo pressure myography of resistance arteries to assess for changes in vasoreactivity. Our goal is to develop a simple model of decreased vascular alpha globin expression without concomitant anemia to further elucidate the roles of endothelial alpha globin in normal physiology and in vascular disease conditions.