A Functional Analysis of SNP on Anthracycline-Induced Cardiotoxicity in a Uniform Genetic Background Using CRISPR/Cas9 Mediated Single-Base-Edited iPSCs

[Introduction]

Anthracycline-induced cardiotoxicity (ACT) has been a major problem in leukemia therapy, and reported to be associated with several candidates of single nucleotide polymorphisms (SNPs). C242T polymorphism (rs4673) in the cytochrome b-245 alpha chain (CYBA) gene, which encodes superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase light chain subunit, is focused on as one of ACT related SNPs. Several clinical studies indicated that rs4673 T allele increases the risk of ACT, however, it is unclear how rs4673 affects ACT. Here, we established a series of induced pluripotent stem cells (iPSCs) with a genotype of C/C, T/T or C/T of rs4673 in a uniform genetic background by using CRISPR/ Cas9, and evaluated the effect of each SNP genotype on ACT in vitro.

[Methods and Results]

To edit the SNP site using CRISPR/Cas9, three guide RNAs (gRNAs) were designed to induce double strand break (DSB) near the target site. Cas9 nuclease and gRNA expression vectors were transfected into HEK293T cells using jetPEI (Polyplus-transfection SAS), and the genome editing activity was assessed by T7 endonucleaseⅠ(T7E1) assay to select more efficient gRNA for cleavage near the target site. The indel frequencies of sgRNAs 1,2 and 3 were 29.4%, 25.6%, and 18.6%, respectively. Furthermore, oligo-DNA (100 nt) to edit single-base in the SNP site was prepared. To generate Cas9/gRNA ribonucleoprotein (RNP) complexes, 1 μg of Cas9 protein and 0.2 μg of gRNA molecules were mixed. RNP complexes and 50 pmol of oligo-DNA were electroporated into human iPSC 201B7cells (Riken BioResource Center) using Neon electroporation device (Invitrogen),single-base-edited iPSCs (C/C and T/T from C/T genotype in rs4673) were established by homology-directed repair (HDR). Successful HDR events were verified by Sanger sequencing. These established iPSCs were differentiated into cardiomyocytes using PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) in vitro. To facilitate nascent mesoderm induction, TBX6 was expressed transiently by a tetracycline-inducible lentiviral vector. Differentiated cardiomyocytes were revealed to be contracting and be positive for NK2 transcription factor related locus 5 (NKX2-5) and troponin T2 (TNNT2) by immunofluorescence staining.In order to evaluate the ACT depending on the difference of single base in rs4673, doxorubicin (DOX)was administered to each edited iPSC-derived cardiomyocyte. NADP⁺/NADPH before and after administration of DOX (1μM) in C/C cardiomyocytes was 1±0.18 and 0.93±0.14, respectively. On the other hand, it was 1±0.07 and 1.29±0.03 in T/T cardiomyocytes (p<0.05, Welch test). T/T cardiomyocytes activated NADP⁺/NADPH cycling after administration of DOX, while C/C cardiomyocytes did not.

[Conclusion]

SNPs are a factor of genetic diversity among individuals, and have been revealed to be associated with various traits and diseases by Genome-Wide Association Study (GWAS). However, the diverse genetic backgrounds often hamper to analyze the influence of each SNP on the trait. In this study, we analyzed the effects of rs4673 on ACT in a uniform genetic background by using CRISPR/Cas9 edited iPSCs. Our study showed that T/T cardiomyocytes activated NADP⁺/NADPH cycling after administration of DOX. These results suggest that rs4673T allele may increase the risk of ACT. Genome editing mediated single-base-alteration will provide us a novel system for functional analysis of SNPs.