A Perfect Moral Storm
CRISPR/Cas9 gene editing can be used to eliminate entire chromosomes: a Q&A
, using CRISPR/Cas9 gene editing techniques to delete entire chromosomes, which may have implications for future therapeutics and our understanding of aneuploidy in tumorigenesis.
Why is eliminating an entire sex chromosome something you’ve been working on?
As is well known, CRISPR/Cas9 is an effective tool for gene editing, Cas Ethical Implications , so far only a few genes have been assessed for biological function in targeted knockout mice. Therefore, besides Y chromosome single-copy genes, we also tested whether Y chromosome genes with multiple copies could be efficiently deleted. To our surprise, we found that deletion of multiple-copy genes,
, resulted in sex reversal of male embryos. Experimental data revealed that the sex reversal was caused by an entire Y chromosome deletion, rather than the knockout of the multiple-copy genes. Thus, an entire chromosome, including sex chromosome X and Y, as well as an autosome, could be deleted by multiple DNA cleavages on the specific chromosome, induced by a sgRNA that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site.
With the advent of CRISPR/Cas9, scientists have been trying to apply this technique to multiple fields, including gene deletion, gene insertion, large fragment deletion and chromosome translocation. However, whether an entire chromosome could be deleted via CRISPR/Cas9 is an intriguing question. Previous studies showed that targeted chromosome elimination could be achieved by insertion of oppositely oriented loxP sites into a targeted chromosome followed by Cre-mediated sister-chromatid recombination, or by insertion of a TKNEO transgene into one copy of targeted chromosome followed by drug selection of chromosome-deletion clones via spontaneous chromosome loss. Both of these approaches require two-step manipulation and have a low yield of obtaining chromosome-deleted cells, thus unsuitable for
studies. By contrast, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes. Cas Ethical Implications , and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.
What are the clinical implications of this technique? Could you highlight a few specific conditions that this could prevent in the future?
Aneuploidy is a human genetic disorder related to the addition or deletion of a chromosome, leading to significant morbidity and mortality during infancy or childhood, including Down’s syndrome (an extra 21), Klinefelter Syndrome (an extra X) or XYY syndrome. Using CRISPR/Cas9 – mediated targeted chromosome elimination, an extra chromosome could be selectively eliminated in cultured cells, embryos, and, more importantly, tissues
, providing a potential therapeutic approach for aneuploidy diseases. However, when one of two homologous X chromosome was deleted by this approach, we found that the remaining X chromosome was also mutated. We believe these mutations in the remaining one (XXY and XYY syndrome) or two chromosomes (Down’s syndrome) could be avoided by using sgRNAs that target only one of the two or one of the three homologous chromosomes, we found that the remaining X chromosome was also mutated. We believe these mutations in the remaining one (XXY and XYY syndrome) or two chromosomes (Down’s syndrome) could be avoided by using sgRNAs that target only one of the two or one of the three homologous chromosomes Cas Ethical Implications
Could you describe how you achieved the elimination of a whole chromosome, what were the main challenges?
CRISPR/Cas9 is like a molecular scissor, which can specifically cut designated positions in a DNA sequence through the guide of sgRNA. In essence, the chromosome is a series of very long DNA sequences, with repetitive sequences on it. These repetitive sequences come up at particular intervals. By designing sgRNAs targeting these repetitive sequences, CRISPR/Cas9 can break all the repeats in the chromosome. You can image that as a tie being cut hundreds of times, and the chromosome would be destroyed beyond the limits of cell repair. Therefore, the targeted chromosome cannot participate in the process of DNA replication during the cell division, and will disappear in the offspring.
The main challenge of this technique is how to induce multiple DNA cleavages efficiently. The efficiency of chromosome deletion varies among different repeat sequences and it is very hard to predict whether these repetitive sequences “work well”. The other challenge is how to selectively targeted one of two or three chromosomes.
What are the ethical implications of gene editing techniques like CRISPR?
As researchers in the field of gene-editing, we all believe that this technology will play an important role in therapeutic treatment. Meanwhile, we fully understand the ethical concerns of this technique in society today, so we always support and obey ethical guidelines regarding use in editing human germ cell, terrorism, threats to ecological security and other guidelines proposed by specialists in the international conference of stem cells, etc. There are many issues to be solved before using this technique to treat patients, such as targeting efficiency, delivery methods and off-target effects. All technologies require necessary rules to constrain, thus we will communicate with various circles of society to ensure the safety of people and the environment.
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