Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders

Abstract

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.

Keywords: chromosome aneuploidy disorder; chromosome elimination; genome editing; iPSC reprogramming.

Figure 1

Schematic overview of elimination of extra chromosome 21 (chr 21) using genome editing technology. (A) Integration of the GFP (EGFP) and HSV-tk gene cassette surrounded by two inverted loxP sites on the homologous arms of chr 21: Cre-dependent recombination between the sister chromatids with inverted loxP generates unstable dicentric and acentric chromosomes for chromosome elimination. (B) Knock-in of the TKneo gene cassette into the amyloid precursor protein (APP) gene exon 3 target locus of one extra copy of chr 21 in Down syndrome (DS)-iPSCs enabled the correction of aneuploidy, followed by positive drug and negative using G418 (neomycin) and GCV (ganciclovir), respectively. (C) Zinc Finger Nuclease (ZFN)-mediated XIST gene knock-in on the Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) gene locus of chr 21 induced Barr body formation to silence the extra copy of chr 21 in DS-iPSCs. (D) CRISPR/Cas9 system targeting the unique repeat sequences introduces multiple DNA double-strand breaks (DSBs) into the target chromosome for deletion of the entire chromosome; XY mouse zygotes injected with Cas9 mRNA and sgRNA to the repeat sequence on the X chromosome for the generation of XO mice in vivo; and DS iPSCs transfected with CRISPR/Cas9 expression vector for multiple cleavages into the extra copy of chr 21 in vitro.

Figure 2

Schematic overview of iPSC reprogramming-mediated chromosome correction. (A) Cell-autonomous correction of the ring chromosome 17 (r(17)) in Miller–Dieker syndrome (MDS) through the loss of abnormal ring chromosome and compensatory uniparental disomy (UPD) mechanism during the reprogramming process. (B) iPSC reprogramming-mediated trisomy-biased chromosome loss corrects the XXY aneuploidy mouse primary fibroblasts from Klinefelter syndrome model mice to rescue the infertility. Euploid XY iPSCs are differentiated into the functional sperms capable to generate the F1 and F2 generation-pups.