Human blastocysts like artificial embryos are called blastoids

An artificial embryo resembling a human blastocyst called a blastoid showing an enveloping layer of extra embryonic cells, a blastula-like cavity, ectoderm cells (green, giving rise to the future embryo) and hypoblast cells (red, giving rise to the future amnion). iMiGSeq for mtDNA sequencing in a single blastoid was used to model the dynamics of mtDNA mutations during human embryonic development. Credit: © 2023 KAUST; Mo me

A high-throughput single-cell genome sequencing technology known as iMiGseq has provided new insights into mitochondrial mutations.[{” attribute=””>DNA (mtDNA) and offers a platform for assessing mtDNA editing strategies and genetic diagnosis of embryos prior to their implantation.

The development of a new high-throughput single-cell single-mitochondrial genome sequencing technology, called iMiGseq, has enabled researchers to uncover previously hidden mutations in mitochondrial DNA (mtDNA) that cause maternally inherited diseases. By allowing for complete sequencing of individual mtDNA in single cells, the iMiGseq method has provided a platform for assessing mtDNA editing strategies, genetic diagnosis of embryos prior to implantation, and understanding the links between mtDNA mutations and complex diseases. The technology has also revealed complex patterns of pathogenic mtDNA mutations, including single nucleotide variants and large structural variants, that were undetectable with conventional next-generation sequencing. Additionally, iMiGseq has shown the potential risks of unintended off-target mutations in a mitochondrial genome editing method called mitoTALEN, highlighting the need for more sensitive methods to assess the safety of editing strategies.

An international team of researchers, led by KAUST stem cell biologist Mo Li, has now quantitatively depicted the genetic maps of mtDNA in single human oocytes (immature eggs) and blastoids (stem cell-based synthetic embryos).[1] This revealed the molecular features of rare mtDNA mutations that cause maternally inherited diseases.

Mitochondria, the “power centers” of cells, play a critical role in cellular communication and metabolism. Human mtDNA is a circular genome containing 37 genes, encoding 13 proteins and a non-coding D-loop region. Heterozygous mutations, inherited from egg cells, can cause congenital diseases, such as Leigh syndrome inherited from the mother, and are associated with complex, late-onset diseases.

“Next-generation sequencing has been used to sequence mtDNA and the implicated heterozygous mutations are important contributors to metabolic diseases. However, understanding of mtDNA mutations is still limited by the limitations of conventional sequencing technologies,” says lead author Chongwei Bi.

“Our new iMiGseq method is important because it enables complete sequencing of individual mtDNA in single cells, allowing unbiased, high-throughput baseline resolution analysis of full-length mtDNA,” says Bi. iMiGseq solves several key questions in this field.

Using third-generation nanohole sequencing technology, the researchers characterized mtDNA heteroplasmy in single cells and described the genetic features of mtDNA in single oocytes. They examined mtDNA in iPS cells derived from patients with Leigh syndrome or neuropathy, ataxia or retinitis pigmentosa (NARP). This revealed complex patterns of disease-causing mtDNA mutations, including single nucleotide variants and large structural variants. “We were able to detect rare mutations with frequencies well below the traditional detection threshold of 1 percent,” says Mo Li.

In another experiment using the new technology, iMiGseq revealed the potential risks of large, unexpected increases in the frequency of off-target mutations, known as heteroplasmy, in a mitochondrial genome-editing method called mitoTALEN — a genome-editing tool that cuts specific sequences in mitochondrial DNA. It is used to interrupt a mutation that causes mitochondrial encephalopathy and stroke-like seizure syndrome in a patient’s iPS cells.

“This highlights the advantages of full-length mtDNA haplotype analysis for understanding heterozygous mitochondrial DNA alteration; other distant mtDNA genetic variants may be inadvertently affected by genetically linked disease-related mutation editing, and ultra-sensitive methods are needed to assess the safety of editing strategies.” as he tells me.

The researchers also used iMiGseq to analyze individual human eggs from healthy donors and individual human blastoids, and artificial embryos made from stem cells, to identify rare mutations that cannot be detected using conventional next-generation sequencing. These heterozygous low-level mutations, potentially inherited through the female germline, are associated with mitochondrial diseases and cancer.[2]

The iMiGseq method provides a novel means to accurately image complete mtDNA haplotypes in single cells, providing an ideal platform to elucidate the cause of diseases associated with mitochondrial mutations, assess the safety of different mtDNA editing strategies and uncover links between mtDNA mutations, aging and complex disease progression.


  1. “Quantitative Analyzed Haplotype Analysis of Mitochondrial DNA Heterologous in Human Single Oocyte, Blastoid, and Pluripotent Stem Cells” by Chongwei Bi, Lin Wang, Yong Fan, Baolei Yuan, Samhan Alsolami, Yingzi Zhang, Pu-Yao Zhang, Yanyi Huang, Yang Yu, Juan Carlos Izpisua Belmonte and Mo Li, April 4, 2023, Available here. Nucleic acid research.
    DOI: 10.1093/nar/gkad209
  2. “Single full-length mtDNA sequencing by iMiGseq reveals unexpected heterologous mutations in mtDNA editing” by Chongwei Bi, Lin Wang, Yong Fan, Baolei Yuan, Gerardo Ramos-Mandujano, Yingzi Zhang, Samhan Alsolami, Xuan Zhou, Jincheng Wang, Yanjiao Shaw, Pradeep Reddy, Bo-Yao Zhang, Yanni Huang, Yang Yu, Juan Carlos Izpisua Belmonte and Mo Li March 31, 2023 Nucleic acid research.
    DOI: 10.1093/nar/gkad208

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