The genetics of human infertility by functional interrogation of SNPs in mice

Abstract

Infertility is a prevalent health issue, affecting ∼15% of couples of childbearing age. Nearly one-half of idiopathic infertility cases are thought to have a genetic basis, but the underlying causes are largely unknown. Traditional methods for studying inheritance, such as genome-wide association studies and linkage analyses, have been confounded by the genetic and phenotypic complexity of reproductive processes. Here we describe an association-and linkagefree approach to identify segregating infertility alleles, in which CRISPR/Cas9 genome editing is used to model putatively deleterious nonsynonymous SNPs (nsSNPs) in the mouse orthologs of fertility genes. Mice bearing "humanized" alleles of four essential meiosis genes, each predicted to be deleterious by most of the commonly used algorithms for analyzing functional SNP consequences, were examined for fertility and reproductive defects. Only a Cdk2 allele mimicking SNP rs3087335, which alters an inhibitory WEE1 protein kinase phosphorylation site, caused infertility and revealed a novel function in regulating spermatogonial stem cell maintenance. Our data indicate that segregating infertility alleles exist in human populations. Furthermore, whereas computational prediction of SNP effects is useful for identifying candidate causal mutations for diverse diseases, this study underscores the need for in vivo functional evaluation of physiological consequences. This approach can revolutionize personalized reproductive genetics by establishing a permanent reference of benign vs. infertile alleles.CRISPR/Cas9 | meiosis | spermatogenesis | cyclin | genome editing

Functional study of a novel missense single‐nucleotide variant of NUP107 in two daughters of Mexican origin with premature ovarian insufficiency

“…Missense variants, which result in single or few nucleotide changes that convert one amino acid to another are much more common, but more difficult to functionally assess. Recent studies using CRISPR/Cas9 suggest that introduction of human variants into mice reveals functionally significant missense variants (Singh & Schimenti, ). Unlike our affected daughters that were infertile and had small ovaries, Nup107 R356C female mice were subfertile and ovaries were not hypoplastic.…”

Genome editing for the reproduction and remedy of human diseases in mice

“…Singh et al [18] tried to identify infertility-causing singlenucleotide polymorphisms (SNPs) involving meiosisassociated genes in humans through generation of mice with single-nucleotide substitutions corresponding to the human SNPs by microinjection of sgRNA, Cas9 mRNA, and ssODN into fertilized eggs. The candidate human SNPs, rs3087335 (CDK2), rs63750447 (MLH1), rs61735519 (SMC1B), and rs147871035 (TEX15), were selected based on the criteria of minor alleles in humans, altering evolutionarily conserved amino acids and changes being predicted as functionally deleterious by multiple algorithms.…”

Functional interrogation of non-coding DNA through CRISPR genome editing

“…Furthermore, this approach was able to achieve HDR-mediated SNP correction in repopulating hematopoietic stem cells, a quiescent cell type traditionally resistant to HDR [146]. In a similar fashion, one study created four different SNP knock-in mice to examine the effect of these SNPs that had previously been implicated in human infertility [156] while another study created knock-in of two disease risk-associated SNPs implicated for lipid levels both in vitro and in vivo [157]. Finally, HDR approaches have also been used to engender genomic deletions as an alternative to dual sgRNA with NHEJ repair as discussed above.…”